Compositions and methods useful for culturing differentiable cells

ABSTRACT

The present invention relates to cell culture methods and compositions that are essentially serum-free and comprise a basal salt nutrient solution and an ErbB3 ligand.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 11/678,487,filed 23 Feb. 2007, which claims priority to U.S. Ser. No. 60/776,113,filed 23 Feb. 2006, both of which are incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Part of the work performed during development of this invention utilizedU.S. Government funds from National Institutes of Health Grant No. 5 R24RR021313-05. The U.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to cell culture methods and compositionsthat are essentially serum-free and comprise a basal salt nutrientsolution and an ErbB3 ligand.

2. Background of the Invention

Human pluripotent cells offer unique opportunities for investigatingearly stages of human development as well as for therapeuticintervention in several disease states, such as diabetes mellitus andParkinson's disease. For example, the use of insulin-producing β-cellsderived from human embryonic stem cells (hESCs) would offer a vastimprovement over current cell therapy procedures that utilize cells fromdonor pancreases. Currently cell therapy treatments for diabetesmellitus, which utilize cells from donor pancreases, are limited by thescarcity of high quality islet cells needed for transplant. Cell therapyfor a single Type I diabetic patient requires a transplant ofapproximately 8×10⁸ pancreatic islet cells (Shapiro et al., 2000, N EnglJ Med 343:230-238; Shapiro et al., 2001a, Best Pract Res Clin EndocrinolMetab 15:241-264; Shapiro et al., 2001, British Medical Journal322:861). As such, at least two healthy donor organs are required toobtain sufficient islet cells for a successful transplant.

Embryonic stem (ES) cells thus represent a powerful model system for theinvestigation of mechanisms underlying pluripotent cell biology anddifferentiation within the early embryo, as well as providingopportunities for genetic manipulation of mammals and resultantcommercial, medical and agricultural applications. Furthermore,appropriate proliferation and differentiation of ES cells canpotentially be used to generate an unlimited source of cells suited totransplantation for treatment of diseases that result from cell damageor dysfunction. Other pluripotent cells and cell lines including earlyprimitive ectoderm-like (EPL) cells as described in International PatentApplication WO 99/53021, in vivo or in vitro derived ICM/epiblast, invivo or in vitro derived primitive ectoderm, primordial germ cells (EGcells), teratocarcinoma cells (EC cells), and pluripotent cells derivedby dedifferentiation or by nuclear transfer will share some or all ofthese properties and applications. International Patent Application WO97/32033 and U.S. Pat. No. 5,453,357 describe pluripotent cellsincluding cells from species other than rodents. Human ES cells havebeen described in International Patent Application WO 00/27995, and inU.S. Pat. No. 6,200,806, and human EG cells have been described inInternational Patent Application WO 98/43679.

The biochemical mechanisms regulating ES cell pluripotency anddifferentiation are very poorly understood. However, the limitedempirical data available (and much anecdotal evidence) suggests that thecontinued maintenance of pluripotent ES cells under in vitro cultureconditions is dependent upon the presence of cytokines and growthfactors present in the extracellular milieu.

While human ESCs offer a source of starting material from which todevelop substantial quantities of high quality differentiated cells forhuman cell therapies, these cells must be obtained and/or cultured inconditions that are compatible with the expected regulatory guidelinesgoverning clinical safety and efficacy. Such guidelines likely willrequire the use of a chemically defined media. The development of suchchemically defined/GMP standard conditions is necessary to facilitatethe use of hESCs and cells derived from hESCs for therapeutic purposesin humans.

In addition, the eventual application of hESC based cell replacementtherapies will require the development of methods that enable largescale culture and differentiation conditions that are compliant withregulatory guidelines. While several groups have reported simplifiedgrowth conditions for hESCs, there are substantial limitations withthese studies. To date, however, the successful isolation, long-termclonal maintenance, genetic manipulation and germ line transmission ofpluripotent cells has generally been difficult.

Most of the cell culture conditions for stem cells still contain serumreplacer (KSR) in the media (Xu et al., 2005 Stem Cells, 23:315-323; Xuet al., 2005 Nature Methods, 2:185-189; Beattie et al., 2005 Stem Cells,23:489-495; Amit et al., 2004 Biol. Reprod., 70:837-845; James et al.,2005 Development, 132:1279-1282). KSR contains a crude fraction ofbovine serum albumin (BSA) rather than a highly purified source. Othershave only performed short-term studies, and therefore it is not clear iftheir conditions would enable the maintenance of pluripotency overextended periods (Sato et al., (2004) Nature Med., 10:55-63; U.S. PatentPublication Nos. 2006/0030042 and 2005/0233446). Others have shownlong-term maintenance of pluripotency in a chemically defined media withFGF2, activin A, and insulin, but the cells were grown on plates thatwere coated with human serum, which was “washed off” before plating ofcells (Vallier et al., 2005 J Cell Sci., 118(Pt 19):4495-509). WhileFGF2 has been a component of all these media, it is not clear if it isan absolute necessity, particularly as in some formulations it isnecessary to use it at a high concentration (up to 100 ng/ml, Xu et al.,2005 Nature Methods, 2:185-189).

Furthermore, all of these groups have either included insulin in theirmedia at μg/ml levels, or have insulin present due to the use of KSR.Insulin is typically considered to function in glucose metabolism and“cell survival” signaling via binding to the insulin receptor. At levelsabove physiological concentrations, however, insulin can also bind tothe IGF1 receptor with a lower efficiency and confer classical growthfactor activity through the PI3 Kinase/AKT pathway. Thepresence/requirement for such high levels of insulin (μg/ml levels) inKSR or these other media conditions suggests that the major activity iselicited via binding to the IGF1 receptor, which is expressed by hESCs(Sperger et al., 2003 PNAS, 100(23):13350-13355). Others have noted theexpression of a full complement of IGF1R and intracellular signalingpathway members in hESCs, which is likely to signify the functionalactivity of this pathway (Miura et al., 2004 Aging Cell, 3:333-343).Insulin or IGF1 may elicit a major signal required for the self-renewalof hESCs, as is suggested by the fact that all conditions developed thusfar for the culture of hESC contain either insulin, insulin provided byKSR, or IGF1 provided by serum. In support of this concept, it has beenshown that if PI3 Kinase is inhibited in hESC cultures, the cellsdifferentiate (D'Amour et al., 2005 Nat. Biotechnol., 23(12):1534-41;McLean et al., 2007 Stem Cells 25:29-38).

A recent publication outlines a humanized, defined media for hESCs(Ludwig et al., Nature Biotechnology, published online Jan. 1, 2006,doi:10.1038/nbt1177). This recent formulation, however, includes severalfactors that are suggested to influence the proliferation of hESCs,including FGF2, TGFβ, LiCl, γ-aminobutyric acid and pipecolic acid. Itis noted that this recently defined cell culture medium also containsinsulin.

The EGF growth factor family has at least 14 members, including, but notlimited to, EGF, TGFβ, heparin binding-EGF (hb-EGF), neuregulin-β (alsonamed heregulin-β (HRG-β), glial growth factor and others), HRG-α,amphiregulin, betacellulin, and epiregulin. All these growth factorscontain an EGF domain and are typically first expressed as transmembraneproteins that are processed by metalloproteinase (specifically, ADAM)proteins to generate soluble ectodomain growth factors. EGF familymembers interact with either homo- or hetero-dimers of the ErbB1, 2, 3and 4 cell surface receptors with different affinities (Jones et al.,FEBS Lett, 1999, 447:227-231). EGF, TGFα and hbEGF bind ErbB1/1 (EGFR)homodimers and ErbB1/2 heterodimers at high affinity (1-100 nM range),whereas HRG-β binds ErbB3 and ErbB4 at very high affinity (<1 nM range).Activated ErbB receptors signal through the PI3 Kinase/AKT pathway andalso the MAPK pathway. ErbB2 and ErbB3 are amongst the most highlyexpressed growth factor receptors in hESCs (Sperger et al., 2003 PNAS,100(23):13350-13355) and HRG-β has been shown previously to support theexpansion of mouse primordial germ cells (Toyoda-Ohno et al., 1999 Dev.Biol., 215(2):399-406). Furthermore, overexpression and subsequentinappropriate activation of ErbB2 is associated with tumorigenesis (Neveet al., 2001 Ann. Oncol., 12 Suppl 1:S9-13; Zhou & Hung, 2003 Semin.Oncol., 30(5 Suppl 16):38-48; Yarden, 2001 Oncology, 61 Suppl 2:1-13).Human ErbB2 (Chromosome 17q), and ErbB3 (Chromosome 12q) are present onchromosomes that have been observed to accumulate as trisomies in somehESCs (Draper et al., 2004 Nat. Biotechnol., 22(1):53-4; Cowan et al.,2004 N Engl. J. Med., 350(13):1353-6; Brimble et al., 2004 Stem CellsDev., 13(6):585-97; Maitra et al., 2005 Nat. Genet. 37(10):1099-103;Mitalipova et al., 2005 Nat. Biotechnol. 23(1): 19-20; Draper et al.,2004 Stem Cells Dev., 13(4):325-36; Ludwig et al., Nature Biotechnology,published online Jan. 1, 2006, doi: 10.1038/nbt1177).

ErbB2 and ErbB3 (Brown et al., 2004 Biol. Reprod., 71:2003-2011;Salas-Vidal & Lomeli, 2004, Dev Biol., 265:75-89) are expressed in themouse blastocyst, although not specifically restricted to the inner cellmass (ICM), and ErbB1, EGF and TGFβ are expressed in the humanblastocyst (Chia et al., 1995 Development, 1221(2):299-307). HB-EGF hasproliferative effects in human IVF blastocyst culture (Martin et al.,1998 Hum. Reprod., 13(6): 1645-52; Sargent et al., 1998 Hum. Reprod. 13Suppl 4:239-48), and modest additional effects on mouse ES cells grownin 15% serum (Heo et al., 2005, Am. J. Phy. Cell Physiol., in press).Pre- and early post-implantation development does not appear to beaffected in ErbB2−/−, ErbB3−/−, Neuregulin1−/− (Britsch et al., 1998Genes Dev., 12:1825-36), ADAM17−/− (Peschon, et al., 1998 Science, 282:1281-1284) and ADAM19−/− (Horiuchi, 2005 Dev. Biol., 283(2):459-71) nullembryos. Therefore, the importance of signaling through the ErbBreceptor family in hESCs is, up to now, unclear.

Neuregulin-1 (NRG1) is a large gene that exhibits multiple splicing andprotein processing variants. This generates a large number of proteinisoforms, which are referred to herein collectively as neuregulin.Neuregulin is predominantly expressed as a cell surface transmembraneprotein. The extracellular region contains an immunoglobulin-likedomain, a carbohydrate modified region and the EGF domain. NRG1expression isoforms have been reviewed previously (Falls, 2003 Exp. CellRes., 284: 14-30). The cell membrane metalloproteases ADAM17 and ADAM19have been shown to process the transmembrane form(s) of neuregulin-1 tosoluble neuregulin/heregulin. HRG-α and -β are the cleaved ectodomainsof neuregulin, containing the EGF and other domains. As the EGF domainis responsible for binding and activation of the ErbB receptors, arecombinant molecule containing only this domain can exhibit essentiallyall of the soluble growth factor effects of this protein (Jones et al.,1999 FEBS Lett., 447:227-231). Also, there are processed transmembraneisoforms of neuregulin that are thought to trigger juxtacrine signalingin adjacent cells via interaction of the EGF domain with ErbB receptors.

An important development in the progression of hESC research towardmaintaining pluripotency in culture will be the elucidation of media andcell culture conditions that are compatible with the expected regulatoryguidelines governing clinical safety and efficacy. While the bestoutcome would be the availability of chemically defined media for hESCs,components that are not chemically defined would be acceptable if theywere produced to GMP standard. There is a need, therefore, to identifymethods and compositions for the culture and stabilization of apopulation of pluripotent stem cells that are able to be used fortherapeutic purposes, wherein the culture compositions are definedand/or produced to GMP standard.

SUMMARY OF THE INVENTION

The invention relates to compositions comprising a basal salt nutrientsolution and an ErbB3 ligand, with the compositions being essentiallyfree of serum.

The invention also relates to compositions comprising a basal saltnutrient solution and a means for stimulating ErbB2-directed tyrosinekinase activity in differentiable cells.

The invention relates to methods of culturing differentiable cells, withthe methods comprising plating the differentiable cells on a cellculture surface, providing a basal salt nutrient solution to thedifferentiable cells and providing a ligand that specifically bindsErbB3.

The invention relates to methods of culturing differentiable cells, withthe methods comprising plating the differentiable cells on a cellculture surface and providing a basal salt nutrient solution to thedifferentiable cells and a means for stimulating ErbB2-directed tyrosinekinase activity in the differentiable cells.

The invention also relates to methods of culturing differentiable cells,with the methods comprising providing a digest solution to a layer ofdifferentiable cells that are contained in a culture chamber prior todigestion, where the digestion breaks apart the layer of cells intosingle cells. After digestion, the single cells are placed into a newtissue culture chamber with a differentiable cell culture solution,wherein the differentiable cell culture solution comprises a basal saltnutrient solution and an ErbB3 ligand. Once cultured, the singledifferentiable cells are placed in conditions that permit growth anddivision of the single cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts real time RT-PCR expression analysis of ADAM19,Neuregulin1, and ErbB1-3 in BG01v grown in defined conditions (8 ng/mlFGF2, 100 ng/ml LR-IGF1, 1 ng/ml Activin A). GAPDH and OCT4 controlreactions are indicated.

FIG. 2 depicts the inhibition of proliferation of BG01v cells usingAG879. BG01v cells were plated in 6-well trays and exposed to DMSO (A),50 nM-20 μM AG1478 (B), or 100 mM-20 μM AG879 (C) 24 hours afterplating. After 5 days in culture, the cultures were fixed and stainedfor alkaline phosphatase activity. AG1478 did not appear to affectproliferation at these concentrations (20 μM shown in B), but AG879substantially slowed cell growth at 5 μM (C).

FIG. 3 depicts the morphology of BG01v cells cultured in DC-HAIF, whichis defined culture media containing 10 ng/ml HRG-β, 10 ng/ml Activin A,200 ng/ml LR-IGF1 and 8 ng/ml FGF2 (A and B), and in defined culturemedia (DC) containing 10 ng/ml HRG-β, 10 ng/ml Activin A, and 200 ng/mlLR-IGF1 (C and D).

FIG. 4 depicts the expression of ADAM19, Neuregulin1, and ErbB1-4 byRT-PCR in mouse ES cells (A) and MEFs (B).

FIG. 5 depicts the inhibition of ErbB1 and ErbB2 signaling in mouse EScells. 2×10⁵ Mouse R1 ES cells were plated on 1:1000 MATRIGEL™ in 10%FBS, 10% KSR with 1000 U/ml mouse LIF (ESGRO). The following day, DMSO(carrier control), 1-50 μM AG1478, or 1-50 μM AG879 was added with freshmedium. The cultures were fixed on day 8, and stained for alkalinephosphatase activity. DMSO (A) and 1-50 μM AG1478 (B and C) did notovertly inhibit proliferation. AG879 substantially inhibited cell growthat 50 μM (compare D and F) and may have slowed proliferation at 20 μM(E).

FIG. 6 depicts the inhibition of proliferation of BG02 cells grown inconditioned media (CM). (A) 50 μM AG825 inhibited proliferation of BG02hESCs growing in CM. (B) AG825 inhibits ErbB2 Y1248 phosphorylation inhESCs. (C) Colony counting of serial passaging of CyT49 hESCs indifferent combinations of growth factors. (D) Cell counting analysis ofthe role of IGF1 and HRG in hESC proliferation using BG02 cells (left).(E) OCT4/DAPI immunostaining of a duplicate repeated experimentdemonstrated that IGF1 and HRG significantly increased the proportion ofOCT4⁺ cells compared to ActA/FGF2 conditions. (F) RTK blotting analysisof BG01 DC-HAIF hESCs starved of growth factors overnight; starved, thenpulsed with DC-HAIF for 15 minutes; or steady-state cultures are shown(left). The mean and range of normalized relative intensity is plotted(right).

FIG. 7 depicts mouse ES cells grown in defined conditions with differentgrowth factor combinations. (A) shows the scoring of AP⁺ colonies after2×10⁵ cells were grown in different growth factor combinations for 8days. (B-G) show 4× magnification images of AP⁺ colonies grown indifferent growth factor combinations.

FIG. 8 depicts the characterization of human ES cells that aremaintained in DC-HAIF medium. (A) Analysis of teratomas from BG02DC-HAIF p25 cells demonstrated pluripotent differentiation potential toectoderm, mesoderm and endoderm. (B) Immunostaining of BG02 cellscultured in 15% FCS/5% KSR that have differentiated. (C) Venn diagram ofthe distribution of transcripts detected using high density IlluminaSentrix Human-6 Expression Beadchips containing 47,296 transcript probesin BG02 cells maintained in CM (64 passages) or DC-HAIF (10 or 32passages in defined media). (D) Scatterplot analysis demonstrating thatthe transcriptional profile of BG02 DC-HAIF p32 cells is highly similarto that of BG02 cells maintained in CM (top), and was not substantiallyaltered in early and late passage cultures in DC-HAIF (bottom). (E)Hierarchical clustering dendrogram of relative gene expression indifferent populations generated using the Beadstudio software.

FIG. 9 depicts the morphology of cells cultured on humanizedextracellular matrices (ECMs) in the presence of DC-HAIF medium. (A)CyT49 cells (diluted 1:200) growing on growth factor-reduced MATRIGEL™(diluted 1:200). CyT49 cells could also grow on tissue culture dishescoated with (B) whole human serum, (C) human fibronectin, and (D)VITROGRO™.

FIG. 10 depicts the single-cell passaging of human ES cells. (A-D)Staged imaging of BG02 cells after passaging with ACCUTASE™ and platingabout 5×10⁵ cells in a 60 mm culture dish. (A) 1.5 hours after initialplating, showing viable cells adhering to the dish. (B) At 20 hourspost-plating, the large majority of cells have aggregated to form smallcolonies. These colonies expand by proliferation by day 4, post-plating(C), and over the course of 5-6 days to form an epithelial-likemonolayer covering the entire dish (D). (E) Normal male karyotypedemonstrated in a BG02 culture passaged 19 times with ACCUTASE™ inDC-HAIF.

FIG. 11 depicts cell morphology after single cell passaging of human EScells using (A) ACCUTASE™, (B) 0.25% Trypsin/EDTA, (C) TrypLE, or (D)Versene.

FIG. 12 depicts the large-scale growth of human ES cells cultured inDC-HAIF. (A) Flow cytometric analysis of BG02 cells after expansion to>10¹⁰ cells. >85% of cells expressed OCT4, CD9, SSEA-4, TRA-1-81. (B)RT-PCR analysis of expression of markers of pluripotency OCT4, NANOG,REX1, SOX2, UTF1, CRIPTO, FOXD3, TERT and DPPA5. Markers ofdifferentiated lineages, α-fetoprotein (AFP), MSX1 and HAND1 were notdetected. (C) Fluorescence in situ hybridization (FISH) using humanchromosome-specific repeats demonstrated maintenance of normal copynumbers for hChr 12, 17, X and Y.

FIG. 13 depicts the morphology (A) and normal karyotype (B) of hESC BG02cells grown in defined media comprising HRG-β and IGF1, but in theabsence of FGF2 for 7 passages, or >2 months.

FIG. 14 depicts a scatterplot analysis of transcripts from hESCs (BG02)that are maintained in DC-HAIF (32 passages) or DC-HAI (10 passages). Alarge proportion of the expressed transcripts were detected in bothsamples, and transcription was not substantially altered by culturinghESCs in the absence of exogenous FGF2. Correlation coefficients (R²)were generated using all detected transcripts with an expression levelof >0 (all dots), or with transcripts exhibiting a detection confidencelevel of >0.99 (R² select, dots indicated by dashed oval). Angled linesdelineate the mean and limits of a 2-fold difference.

FIG. 15 depicts a hierarchical clustering dendrogram of relative geneexpression in different populations of early and late passage BG02 cellsmaintained in DC-HAIF. Cells clustered tightly (˜0.0075) and retained aclose similarity to BG02 and BG03 cells maintained in conditioned medium(CM) (˜0.037). BG02 cells maintained in DC-HAI also clustered tightlywith the other hESC populations examined. By way of explanation in FIG.15, CM is Conditioned Medium; DC is defined culture medium, DC-HAIF asdefined above; ap is ACCUTASE™ single cell passaging; DC-HAI isidentical to DC-HAIF as defined herein, except without FGF2.

FIG. 16 depicts the morphology and alkaline phsophatase staining of BG02cells cultured in DC-HAIF in 96-well and 384-well plates. (A) Phasecontrast imaging and (B) alkaline phosphatase staining of BG02 cells(10⁴ cells/well) growing in one well of a 96-well plate. (C) Phasecontrast imaging and (D) alkaline phosphatase staining of BG02 cells(10³ cells/well) growing in one well of a 384-well plate.

FIG. 17 depicts dark field images of BG02 grown in DC-HAIF in suspensionculture. Day 2 and day 6 cultures are shown. The images were capturedusing 4× magnification

FIG. 18 depicts the growth rates in adherent and suspension cultures inDC-HAIF. 1×10⁶ BG02 cells were plated into parallel wells in adherentand suspension culture and cell counts were performed on days 1-6.

FIG. 19 depicts qPCR analysis of suspension and adherent hESCs. BG02cells growing in suspension (S. hESCs) and adherent (hESCs) cultureexhibited comparable levels of OCT4, and lacked SOX17 expression.Adherent cells differentiated to definitive endoderm (DE), andsuspension hESCs differentiated to definitive endoderm in suspension (S.DE d3), both exhibited the expected marked downregulation of OCT4 andupregulation of SOX17 expression

FIG. 20 depicts the enhancement of hESC aggregation in the presence ofY27632 in suspension culture. 2×10⁶ BG02 cells were seeded in 3 mlDC-HAIF or DC-HAIF+Y27632, in 6-well trays, in an incubator on arotating platform at 100 rpm. Images of aggregates were captured on days1 and 3.

FIG. 21 depicts RT-PCR analysis of suspension aggregates in the presenceof Y27632. RT-PCR was performed on the expanded cultures to assessexpression of markers of pluripotency. Expression of OCT4, NANOG, REX1,SOX2, UTF1, CRIPTO, FOXD3, TERT AND DPPA5 were detected, whereas markersof differentiated lineages AFP, MSX1 and HAND1 were not detected.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise noted, the terms used herein are to be understoodaccording to conventional usage by those of ordinary skill in therelevant art. In addition to the definitions of terms provided below,definitions of common terms in molecular biology may also be found inRieger et al., 1991 Glossary of genetics: classical and molecular, 5thEd., Berlin: Springer-Verlag; and in Current Protocols in MolecularBiology, F. M. Ausubel et al., Eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(1998 Supplement). It is to be understood that as used in thespecification and in the claims, “a” or “an” can mean one or more,depending upon the context in which it is used. Thus, for example,reference to “a cell” can mean that at least one cell can be utilized.

As used herein, the term “contacting” (i.e., contacting a cell e.g., adifferentiable cell, with a compound) is intended to include incubatingthe compound and the cell together in vitro (e.g., adding the compoundto cells in culture). The term “contacting” is not intended to includethe in vivo exposure of cells to a defined cell medium comprising anErbB3 ligand, and optionally, a member of the TGF-β family, that mayoccur naturally in a subject (i.e., exposure that may occur as a resultof a natural physiological process). The step of contacting the cellwith a defined cell medium comprising an ErbB3 ligand, and optionally, amember of the TGF-β family, can be conducted in any suitable manner. Forexample, the cells may be treated in adherent culture, or in suspensionculture. It is understood that the cells contacted with the definedmedium can be further treated with a cell differentiation environment tostabilize the cells, or to differentiate the cells.

As used herein, the term “differentiate” refers to the production of acell type that is more differentiated than the cell type from which itis derived. The term therefore encompasses cell types that are partiallyand terminally differentiated.

In certain embodiments of the present invention, the term “enriched”refers to a cell culture that contains more than approximately 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the desired cell lineage.

As used herein, the term “effective amount” of a compound refers to thatconcentration of the compound that is sufficient in the presence of theremaining components of the defined medium to effect the stabilizationof the differentiable cell in culture for greater than one month in theabsence of a feeder cell and in the absence of serum or serumreplacement. This concentration is readily determined by one of ordinaryskill in the art.

As used herein, the term “express” refers to the transcription of apolynucleotide or translation of a polypeptide in a cell, such thatlevels of the molecule are measurably higher in a cell that expressesthe molecule than they are in a cell that does not express the molecule.Methods to measure the expression of a molecule are well known to thoseof ordinary skill in the art, and include without limitation, Northernblotting, RT-PCR, in situ hybridization, Western blotting, andimmunostaining.

As used herein when referring to a cell, cell line, cell culture orpopulation of cells, the term “isolated” refers to being substantiallyseparated from the natural source of the cells such that the cell, cellline, cell culture, or population of cells are capable of being culturedin vitro. In addition, the term “isolating” is used to refer to thephysical selection of one or more cells out of a group of two or morecells, wherein the cells are selected based on cell morphology and/orthe expression of various markers.

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein. However, before the presentcompositions and methods are disclosed and described, it is to beunderstood that this invention is not limited to specific nucleic acids,specific polypeptides, specific cell types, specific host cells,specific conditions, or specific methods, etc., as such may, of course,vary, and the numerous modifications and variations therein will beapparent to those skilled in the art.

Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described inSambrook et al., 1989 Molecular Cloning, Second Edition, Cold SpringHarbor Laboratory, Plainview, N.Y.; Maniatis et al., 1982 MolecularCloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (Ed.) 1993Meth. Enzymol. 218, Part I; Wu (Ed.) 1979 Meth. Enzymol. 68; Wu et al.,(Eds.) 1983 Meth. Enzymol. 100 and 101; Grossman and Moldave (Eds.) 1980Meth. Enzymol. 65; Miller (ed.) 1972 Experiments in Molecular Genetics,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old andPrimrose, 1981 Principles of Gene Manipulation, University of CaliforniaPress, Berkeley; Schleif and Wensink, 1982 Practical Methods inMolecular Biology; Glover (Ed.) 1985 DNA Cloning Vol. I and II, IRLPress, Oxford, UK; Hames and Higgins (Eds.) 1985 Nucleic AcidHybridization, IRL Press, Oxford, UK; and Setlow and Hollaender 1979Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press,New York. Abbreviations and nomenclature, where employed, are deemedstandard in the field and commonly used in professional journals such asthose cited herein.

The invention relates to compositions and methods comprising a basalsalt nutrient solution and an effective amount of an ErbB3 ligand, withthe compositions being essentially free of serum. The compositions andmethods of the present invention are useful for culturing cells, inparticular, differentiable cells. It is understood that at differentpoints during culturing the differentiable cells, various components maybe added to the cell culture such that the medium can contain componentsother than those described herein. It is, however, contemplated that atleast at one point during the preparation of the culture, or during theculture of the differentiable cells, the defined medium comprises abasal salt nutrient solution and a means for activating ErbB2-directedtyrosine kinase.

As used herein, the term “differentiable cell” is used to describe acell or population of cells that can differentiate into at leastpartially mature cells, or that can participate in the differentiationof cells, e.g., fuse with other cells, that can differentiate into atleast partially mature cells. As used herein, “partially mature cells”are cells that exhibit at least one characteristic of the phenotype,such as morphology or protein expression, of a mature cell from the sameorgan or tissue. For example, a normal, mature hepatocyte typicallyexpresses such proteins as albumin, fibrinogen, alpha-1-antitrypsin,prothrombin clotting factors, transferrin, and detoxification enzymessuch as the cytochrome P-450s, among others. Thus, as defined in thepresent invention, a “partially mature hepatocyte” may express albuminor another one or more proteins, or begin to take the appearance orfunction of a normal, mature hepatocyte. Additionally, a “partiallymature pancreatic beta cell” may produce or express the proinsulinprotein, among others. The ability of the cells to differentiate into atleast partially mature cells will not be dependent upon recombinantengineering techniques, such as transfection, though the cells may, ofcourse, be genetically engineered.

The invention contemplates compositions and methods useful fordifferentiable cells, regardless of their source or of their plasticity.The “plasticity” of a cell is used herein roughly as it is in the art.Namely, the plasticity of a cell refers to a cell's ability todifferentiate into a particular cell type found in tissues or organsfrom an embryo, fetus or developed organism. The “more plastic” a cell,the more tissues into which the cell may be able to differentiate.“Pluripotent cells” include cells and their progeny, which may be ableto differentiate into, or give rise to, pluripotent, multipotent,oligopotent and unipotent cells, and/or several, if not all, of themature or partially mature cell types found in an embryo, fetus ordeveloped organism. “Multipotent cells” include cells and their progeny,which may be able to differentiate into, or give rise to, multipotent,oligopotent and unipotent progenitor cells, and/or one or more mature orpartially mature cell types, except that the mature or partially maturecell types derived from multipotent cells are limited to cells of aparticular tissue, organ or organ system. For example, a multipotenthematopoietic progenitor cell and/or its progeny possess the ability todifferentiate into or give rise to one or more types of oligopotentcells, such as myeloid progenitor cells and lymphoid progenitor cells,and also give rise to other mature cellular components normally found inthe blood. “Oligopotent cells” include cells and their progeny whoseability to differentiate into mature or partially mature cells is morerestricted than multipotent cells. Oligopotent cells may, however, stillpossess the ability to differentiate into oligopotent and unipotentcells, and/or one or more mature or partially mature cell types of agiven tissue, organ or organ system. One example of an oligopotent cellis a myeloid progenitor cell, which can ultimately give rise to matureor partially mature erythrocytes, platelets, basophils, eosinophils,neutrophils and monocytes. “Unipotent cells” include cells and theirprogeny that possess the ability to differentiate or give rise to otherunipotent cells and/or one type of mature or partially mature cell type.

Differentiable cells, as used herein, may be pluripotent, multipotent,oligopotent or even unipotent. In certain embodiments of the presentinvention, the differentiable cells are pluripotent differentiablecells. In more specific embodiments, the pluripotent differentiablecells are selected from the group consisting of embryonic stem cells,ICM/epiblast cells, primitive ectoderm cells, primordial germ cells, andteratocarcinoma cells. In one particular embodiment, the differentiablecells are mammalian embryonic stem cells. In a more particularembodiment, the differentiable cells are human embryonic stem cells.

The invention also contemplates differentiable cells from any sourcewithin an animal, provided the cells are differentiable as definedherein. For example, differentiable cells may be harvested from embryos,or any primordial germ layer therein, from placental or chorion tissue,or from more mature tissue such as adult stem cells including, but notlimited to adipose, bone marrow, nervous tissue, mammary tissue, livertissue, pancreas, epithelial, respiratory, gonadal and muscle tissue. Inspecific embodiments, the differentiable cells are embryonic stem cells.In other specific embodiments, the differentiable cells are adult stemcells. In still other specific embodiments, the stem cells areplacental- or chorionic-derived stem cells.

Of course, the invention contemplates using differentiable cells fromany animal capable of generating differentiable cells. The animals fromwhich the differentiable cells are harvested may be vertebrate orinvertebrate, mammalian or non-mammalian, human or non-human. Examplesof animal sources include, but are not limited to, primates, rodents,canines, felines, equines, bovines and porcines.

The differentiable cells of the present invention can be derived usingany method known to those of skill in the art. For example, humanpluripotent cells can be produced using de-differentiation and nucleartransfer methods. Additionally, the human ICM/epiblast cell or theprimitive ectoderm cell used in the present invention can be derived invivo or in vitro. Primitive ectodermal cells may be generated inadherent culture or as cell aggregates in suspension culture, asdescribed in WO 99/53021. Furthermore, the human pluripotent cells canbe passaged using any method known to those of skill in the art,including, manual passaging methods, and bulk passaging methods such asenzymatic or non-enzymatic passaging.

In certain embodiment, when ES cells are utilized, the embryonic stemcells have a normal karyotype, while in other embodiments, the embryonicstem cells have an abnormal karyotype. In one embodiment, a majority ofthe embryonic stem cells have a normal karyotype. It is contemplatedthat greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or greaterthan 95% of metaphases examined will display a normal karyotype.

In another embodiment, a majority of the embryonic stem cells have anabnormal karyotype. It is contemplated that greater than 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90% or greater than 95% of metaphases examinedwill display an abnormal karyotype. In certain embodiments, the abnormalkaryotype is evident after the cells have been cultured for greater than5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 passages. In one specificembodiment, the abnormal karyotype comprises a trisomy of at least oneautosomal chromosome, wherein the autosomal chromosome is selected fromthe group consisting of chromosomes 1, 7, 8, 12, 14, and 17. In anotherembodiment, the abnormal karyotype comprises a trisomy of more than oneautosomal chromosome, wherein at least one of the more than oneautosomal chromosomes is selected from the group consisting ofchromosomes 1, 7, 8, 12, 14, and 17. In one embodiment, the autosomalchromosome is chromosome 12 or 17. In another embodiment, the abnormalkaryotype comprises an additional sex chromosome. In one embodiment, thekaryotype comprises two X chromosomes and one Y chromosome. It is alsocontemplated that translocations of chromosomes may occur, and suchtranslocations are encompassed within the term “abnormal karyotype.”Combinations of the foregoing chromosomal abnormalities and otherchromosomal abnormalities are also encompassed by the invention.

The compositions and methods comprise a basal salt nutrient solution. Asused herein, basal salt nutrient solution refers to a mixture of saltsthat provide cells with water and certain bulk inorganic ions essentialfor normal cell metabolism, maintain intra- and extra-cellular osmoticbalance, provide a carbohydrate as an energy source, and provide abuffering system to maintain the medium within the physiological pHrange. Examples of basal salt nutrient solutions include, but are notlimited to, Dulbecco's Modified Eagle's Medium (DMEM), Minimal EssentialMedium (MEM), Basal Medium Eagle (BME), RPM1 1640, Ham's F-10, Ham'sF-12, α-Minimal Essential Medium (αMEM), Glasgow's Minimal EssentialMedium (G-MEM), and Iscove's Modified Dulbecco's Medium, and mixturesthereof. In one particular embodiment, the basal salt nutrient solutionis an approximately 50:50 mixture of DMEM and Ham's F12.

It is contemplated that the composition can further comprise traceelements. Trace elements can be purchased commercially, for example,from Mediatech. Non-limiting examples of trace elements include but arenot limited to compounds comprising, aluminum, chlorine, sulfate, iron,cadmium, cobalt, chromium, germanium, sodium, potassium, calcium,phosphate and magnesium. Specific example of compounds containing traceelements include but are not limited to, AlCl₃, AgNO₃, Ba(C₂H₃O₂)₂,CdCl₂, CdSO₄, CoCl₂, CrCl₃, Cr₂(SO₄)₃, CuSO₄, ferric citrate, GeO₂, KI,KBr, LI, molybdic acid, MnSO₄, MnCl₂, NaF, Na₂SiO₃, NaVO₃, NH₄VO₃,(NH₄)₆Mo₇O₂₄, NiSO₄, RbCl, selenium, Na₂SeO₃, H₂SeO₃, selenite.2Na,selenomethionone, SnCl₂, ZnSO₄, ZrOCl₂, and mixtures and salts thereof.If selenium, selenite or selenomethionone is present, it is at aconcentration of approximately 0.002 to approximately 0.02 mg/L. Inaddition, hydroxylapatite may also be present.

It is contemplated that amino acids can be added to the defined media.Non-limiting examples of such amino acids are Glycine, L-Alanine,L-Alanyl-L-Glutamine, L-Glutamine/Glutamax, L-Arginine hydrochloride,L-Asparagine-H₂O, L-Aspartic acid, L-Cysteine hydrochloride-H₂O,L-Cystine 2HCl, L-Glutamic Acid, L-Histidine hydrochloride-H₂O,L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine,L-Phenylalanine, L-Proline, L-Hydroxyproline, L-Serine, L-Threonine,L-Tryptophan, L-Tyrosine disodium salt dihydrate, and L-Valine. Incertain embodiments, the amino acid is L-Isoleucine, L-Phenylalanine,L-Proline, L-Hydroxyproline, L-Valine, and mixtures thereof.

It is also contemplated that the defined medium can comprise ascorbicacid. Preferably ascorbic acid is present at an initial concentration ofapproximately 1 mg/L to approximately 1000 mg/L, or from approximately 2mg/L to approximately 500 mg/L, or from approximately 5 mg/L toapproximately 100 mg/L, or from approximately 10 mg/L to approximately100 mg/L or approximately at 50 mg/L.

In addition, the compositions and methods may also comprise othercomponents such as serum albumin, transferrin, L-glutamine, lipids,antibiotics, β-Mercaptoethanol, vitamins, minerals, ATP and similarcomponents may be present. Examples of vitamins that may be presentinclude, but are not limited to vitamins A, B₁, B₂, B₃, B₅, B₆, B₇, B₉,B₁₂, C, D₁, D₂, D₃, D₄, D₅, E, tocotrienols, K₁ and K₂. One of skill inthe art can determine the optimal concentration of minerals, vitamins,ATP, lipids, essential fatty acids, etc., for use in a given culture.The concentration of supplements may, for example, be from about 0.001μM to about 1 mM or more. Specific examples of concentrations at whichthe supplements may be provided include, but are not limited to about0.005 μM, 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1.0 μM, 2.0 μM, 2.5 μM, 3.0M4.0M, 5.0M, 10 μM, 20 μM, 100 μM, etc. In one specific embodiment, thecompositions and methods comprise vitamin B₆ and glutamine. In anotherspecific embodiment, the compositions and methods comprise vitamin C andan iron supplement. In another specific embodiment, the compositions andmethods comprise vitamin K₁ and vitamin A. In another specificembodiment, the compositions and methods comprise vitamin D₃ and ATP. Inanother specific embodiment, the compositions and methods comprisevitamin B₁₂ and transferrin. In another specific embodiment, thecompositions and methods comprise tocotrienols and β-Mercaptoethanol. Inanother specific embodiment, the compositions and methods compriseglutamine and ATP. In another specific embodiment, the compositions andmethods comprise an omega-3 fatty acid and glutamine. In anotherspecific embodiment, the compositions and methods comprise an omega-6fatty acid and vitamin B₁. In another specific embodiment, thecompositions and methods comprise α-linolenic acid and B₂.

The compositions of the present invention are essentially serum free. Asused herein, “essentially serum free” refers to the absence of serum inthe solutions of the present invention. Serum is not an essentialingredient to the compositions and methods of the present invention.Thus, the presence of serum in any of the compositions should only beattributable to impurities, e.g., from the starting materials orresidual serum from the primary cell culture. For example, essentiallyserum free medium or environment can contain less than 10, 9, 8, 7, 6,5, 4, 3, 2, or 1% serum wherein the presently improved bioactivemaintenance capacity of the medium or environment is still observed. Ina specific embodiment of the present invention, the essentially serumfree composition does not contain serum or serum replacement, or onlycontains trace amounts of serum or serum replacement from the isolationof components of the serum or serum replacement that are added to thedefined media.

The compositions and methods of the present invention also comprise ameans for stimulating ErbB2 tyrosine kinase activity withindifferentiable cells. In one specific embodiment, the compositions andmethods of the present invention comprise the presence of at least oneErbB3 ligand. Typically, an ErbB3 ligand will bind the ErbB3 receptorand dimerize with the ErbB2 receptor. The ErbB2 receptor is, in turn,generally responsible for intracellular tyro sine kinase activity withinthe differentiable cell.

As used herein, “ErbB3 ligand” refers to a ligand that binds to ErbB3,which in turn dimerizes to ErbB2, thus activating the tyrosine kinaseactivity of the ErbB2 portion of the ErbB2/ErbB3 heterodimeric receptor.Non-limiting examples of ErbB3 ligands include Neuregulin-1; splicevariants and isoforms of Neuregulin-1, including but not limited toHRG-β, HRG-α, Neu Differentiation Factor (NDF), AcetylcholineReceptor-Inducing Activity (ARIA), Glial Growth Factor 2 (GGF2), andSensory And Motor Neuron-Derived Factor (SMDF); Neuregulin-2; splicevariants and isoforms of Neuregulin-2, including but not limited toNRG2-β; Epiregulin; and Biregulin.

In one embodiment, the means for stimulating ErbB2-directed tyrosinekinase activity comprise at least one ErbB3 ligand that is selected fromthe group consisting of Neuregulin-1, Heregulin-β (HRG-β), Heregulin-α(HRG-α), Neu differentiation factor (NDF), acetylcholinereceptor-inducing activity (ARIA), glial growth factor 2 (GGF2),motor-neuron derived factor (SMDF), Neuregulin-2, Neuregulin-2β(NRG2-β), Epiregulin, Biregulin and variants and functional fragmentsthereof. In another specific embodiment, the compositions and methods ofthe present invention comprise more than one means for stimulatingErbB2-directed tyrosine kinase activity, such as, but not limited to,using more than one ErbB3 ligand.

In a more specific embodiment of the compositions and methods of thepresent invention, the ErbB3 ligand is HRG-β or a variant or functionalfragment thereof. In one embodiment, the species from which the cultureadditive protein, polypeptide or variant or functional fragment thereofderives is the same as the species of cells that are cultured. Forexample, if mouse ES cells are cultured, an HRG-β with an amino acidsequence that is identical to the mus musculus HRG-β sequence can beused as an additive in culture and is considered to be “of the samespecies.” In other embodiments, the species from which the biologicaladditive derives is different from the cells being cultures. Forexample, if mouse ES cells are cultured, an HRG-β with an amino acidsequence that is identical to the human HRG-β sequence from can be usedas an additive in culture and is considered to be “of differentspecies.”

As used herein, a “functional fragment” is a fragment or splice variantof a full length polypeptide that exerts a similar physiological orcellular effect as the full length polypeptide. The biological effect ofthe functional fragment need not be identical in scope or strength asthe full-length polypeptide, so long as a similar physiological orcellular effect is seen. For example, a functional fragment of HRG-β candetectably stimulate ErbB2-directed tyrosine kinase.

As used herein, the term “variant” includes chimeric or fusionpolypeptides, homologs, analogs, orthologs, and paralogs. In addition, avariant of a reference protein or polypeptide is a protein orpolypeptide whose amino acid sequence is at least about 80% identical tothe reference protein or polypeptide. In specific embodiments, thevariant is at least about 85%, 90%, 95%, 95%, 97%, 98%, 99% or even 100%identical to the reference protein or polypeptide. As used herein, theterms “correspond(s) to” and “corresponding to,” as they relate tosequence alignment, are intended to mean enumerated positions within thereference protein or polypeptide, e.g., wild-type human or mouseneuregulin-1, and those positions in the modified protein or polypeptidethat align with the positions on the reference protein or polypeptide.Thus, when the amino acid sequence of a subject protein or polypeptideis aligned with the amino acid sequence of a reference protein orpolypeptide, the sequence that “corresponds to” certain enumeratedpositions of the reference protein or polypeptide sequence are thosethat align with these positions of the reference sequence, but are notnecessarily in these exact numerical positions of the referencesequence. Methods for aligning sequences for determining correspondingamino acids between sequences are described below.

A polypeptide having an amino acid sequence at least, for example, about95% “identical” to a reference an amino acid sequence encoding, forexample TGF-β, is understood to mean that the amino acid sequence of thepolypeptide is identical to the reference sequence except that the aminoacid sequence may include up to about five modifications per each 100amino acids of the reference amino acid sequence encoding the referenceTGF-β. In other words, to obtain a peptide having an amino acid sequenceat least about 95% identical to a reference amino acid sequence, up toabout 5% of the amino acid residues of the reference sequence may bedeleted or substituted with another amino acid or a number of aminoacids up to about 5% of the total amino acids in the reference sequencemay be inserted into the reference sequence. These modifications of thereference sequence may occur at the N-terminus or C-terminus positionsof the reference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among amino acids in thereference sequence or in one or more contiguous groups within thereference sequence.

As used herein, “identity” is a measure of the identity of nucleotidesequences or amino acid sequences compared to a reference nucleotide oramino acid sequence. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. (See, e.g.,Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York (1988); Biocomputing: Informatics And Genome Projects,Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey (1994); von Heinje, G., Sequence Analysis In MolecularBiology, Academic Press (1987); and Sequence Analysis Primer, Gribskov,M. and Devereux, J., eds., M Stockton Press, New York (1991)). Whilethere exist several methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H. & Lipton, D., Siam J Applied Math48:1073 (1988)). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego (1994) and Carillo, H. & Lipton, D., Siam J AppliedMath 48:1073 (1988). Computer programs may also contain methods andalgorithms that calculate identity and similarity. Examples of computerprogram methods to determine identity and similarity between twosequences include, but are not limited to, GCG program package(Devereux, J., et al., Nucleic Acids Research 12(i):387 (1984)), BLASTP,ExPASy, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403(1990)) and FASTDB. Examples of methods to determine identity andsimilarity are discussed in Michaels, G. and Garian, R., CurrentProtocols in Protein Science, Vol 1, John Wiley & Sons, Inc. (2000),which is incorporated by reference. In one embodiment of the presentinvention, the algorithm used to determine identity between two or morepolypeptides is BLASTP.

In another embodiment of the present invention, the algorithm used todetermine identity between two or more polypeptides is FASTDB, which isbased upon the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990), incorporated by reference). In a FASTDB sequence alignment, thequery and subject sequences are amino sequences. The result of sequencealignment is in percent identity. Parameters that may be used in aFASTDB alignment of amino acid sequences to calculate percent identityinclude, but are not limited to: Matrix=PAM, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or thelength of the subject amino sequence, whichever is shorter.

If the subject sequence is shorter or longer than the query sequencebecause of N-terminus or C-terminus additions or deletions, not becauseof internal additions or deletions, a manual correction can be made,because the FASTDB program does not account for N-terminus andC-terminus truncations or additions of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare N- and C-terminus to the reference sequence that are notmatched/aligned, as a percent of the total bases of the query sequence.The results of the FASTDB sequence alignment determinematching/alignment. The alignment percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thiscorrected score can be used for the purposes of determining howalignments “correspond” to each other, as well as percentage identity.Residues of the query (subject) sequences or the reference sequence thatextend past the N- or C-termini of the reference or subject sequence,respectively, may be considered for the purposes of manually adjustingthe percent identity score. That is, residues that are notmatched/aligned with the N- or C-termini of the comparison sequence maybe counted when manually adjusting the percent identity score oralignment numbering.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue reference sequence to determine percent identity. Thedeletion occurs at the N-terminus of the subject sequence and therefore,the FASTDB alignment does not show a match/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 reference sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected.

The invention also provides chimeric or fusion polypeptides. As usedherein, a “chimeric polypeptide” or “fusion polypeptide” comprises atleast a portion of a member of the reference polypeptide operativelylinked to a second, different polypeptide. The second polypeptide has anamino acid sequence corresponding to a polypeptide which is notsubstantially identical to the reference polypeptide, and which isderived from the same or a different organism. With respect to thefusion polypeptide, the term “operatively linked” is intended toindicate that the reference polypeptide and the second polypeptide arefused to each other so that both sequences fulfill the proposed functionattributed to the sequence used. The second polypeptide can be fused tothe N-terminus or C-terminus of the reference polypeptide. For example,in one embodiment, the fusion polypeptide is a GST-IGF-1 fusionpolypeptide in which an IGF-1 sequence is fused to the C-terminus of theGST sequences. Such fusion polypeptides can facilitate the purificationof recombinant polypeptides. In another embodiment, the fusionpolypeptide can contain a heterologous signal sequence at itsN-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of a polypeptide can be increased throughuse of a heterologous signal sequence.

In addition to fragments and fusion polypeptides, the present inventionincludes homologs and analogs of naturally occurring polypeptides.“Homologs” are defined herein as two nucleic acids or polypeptides thathave similar, or “identical,” nucleotide or amino acid sequences,respectively. Homologs include allelic variants, orthologs, paralogs,agonists, and antagonists as defined hereafter. The term “homolog”further encompasses nucleic acid molecules that differ from a referencenucleotide sequence due to degeneracy of the genetic code and thusencode the same polypeptide as that encoded by the reference nucleotidesequence. As used herein, “naturally occurring” refers to a nucleic oramino acid sequence that occurs in nature.

An agonist of a polypeptide can retain substantially the same, or asubset, of the biological activities of the polypeptide. An antagonistof a polypeptide can inhibit one or more of the activities of thenaturally occurring form of the polypeptide.

In another more specific embodiment of the compositions and methods ofthe present invention, the ErbB3 ligand is HRG-β or a variant or afunctional fragment thereof. Additional, non-limiting examples of ErbB3ligands are disclosed in U.S. Pat. Nos. 6,136,558, 6,387,638, and7,063,961, which are incorporated by reference.

Heregulins are generally classified into two major types, alpha andbeta, based on two variant EGF-like domains that differ in theirC-terminal portions. These EGF-like domains, however, are identical inthe spacing of six cysteine residues contained therein. Based on anamino acid sequence comparison, Holmes et al. found that between thefirst and sixth cysteines in the EGF-like domain, HRGs were 45% similarto heparin-binding EGF-like growth factor (HB-EGF), 35% identical toamphiregulin (AR), 32% identical to TGF-α, and 27% identical to EGF.

The 44 kDa neu differentiation factor (NDF) is the rat equivalent ofhuman HRG. Like the HRG polypeptides, NDF has an immunoglobulin (Ig)homology domain followed by an EGF-like domain and lacks a N-terminalsignal peptide. Presently, there are at least six distinct fibroblasticpro-NDFs, classified as either alpha or beta polypeptides, based on thesequences of the EGF-like domains. Isoforms 1 to 4 are characterized onthe basis of a variable stretch between the EGF-like domain andtransmembrane domain. Thus it appears that different NDF isoforms aregenerated by alternative splicing and may perform distincttissue-specific functions. See EP 505 148; WO 93/22424; and WO 94/28133,which are incorporated by reference.

In one embodiment of the present invention, the compositions and methodsare free of exogenous insulin and insulin substitutes. The phrase“exogenous insulin or insulin substitutes” is used herein to indicateinsulin or insulin substitutes that is/are not intentionally added tothe compositions or methods of the present invention. Thus, in certainembodiments of the present invention, the methods and compositions arefree of insulin or insulin substitutes that are intentionally supplied.The compositions or methods may, however, not necessarily be free ofendogenous insulin. As used herein, “endogenous insulin” indicates thatthe cultured cells may be producing insulin of their own accord whencultured according to the methods of the present invention. Endogenousinsulin also may be used to indicate residual impurities from theprimary cell culture or impurities from the starting materials. Inspecific examples, the compositions and methods of the present containless than 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or1 μg/ml of insulin.

As used herein, the term “insulin” refers to the protein, or variant orfragment thereof that binds to the insulin receptor in normalphysiological concentrations and can induce signaling through theinsulin receptor. The term “insulin” encompasses a protein having thepolypeptide sequence of native human insulin, or of other mammalianinsulin, or of any homologs or variants to these sequences.Additionally, the term insulin encompasses polypeptide fragments thatare capable of binding to the insulin receptor to induce signalingthrough the insulin receptor. The term “insulin substitute” refers toany zinc containing compound that may be used in place of insulin togive substantially similar results as insulin. Examples of insulinsubstitutes include, but are not limited to zinc chloride, zinc nitrate,zinc bromide, and zinc sulfate.

To be clear, insulin-like growth factors are not insulin substitutes orhomologs of insulin, as contemplated in the present invention.Accordingly, in another specific embodiment, the compositions andmethods of the present invention comprise the use of at least oneinsulin-like growth factor (IGF) or a variant or a functional fragmentthereof. In another embodiment, the compositions and methods of thepresent invention are free of any exogenous insulin-like growth factors(IGFs). In specific embodiments, the compositions and methods of thepresent invention contain less than 200, 150, 100, 75, 50, 25, 20, 15,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng/ml of IGF-1.

As used herein, the term “activator of IGF-1R” refers to mitogens thatplay a pivotal role in regulating cell proliferation, differentiation,and apoptosis. The effects of an activator of IGF-1R are typicallymediated through IGF-1R, although they can be mediated through otherreceptors. The IGF-1R is also involved in cell transformation induced bytumor virus proteins and oncogene products, and the interaction isregulated by a group of specific binding proteins (IGFBPs). In addition,a large group of IGFBP proteases hydrolyze IGFBPs, resulting in therelease of bound IGFs that then resume their ability to interact withIGF-IR. For the purpose of this invention, the ligands, the receptors,the binding proteins, and the proteases are all considered to beactivators of IGF-1R. In one embodiment, the activator of IGF-1R isIGF-1, or IGF-2. In a further embodiment, the activators of IGF-1R is anIGF-1 analog. Non-limiting examples of IGF-1 analogs includeLongR3-IGF1, Des(1-3)IGF-1, [Arg³]IGF-1, [Ala³¹]IFG-1,Des(2,3)[Ala³¹]IGF-1, [Leu 24]IGF1, Des(2,3)[Leu 24]IGF-1, [Leu60]IGF-1, [Ala³¹][Leu 60]IGF-1, [Leu²⁴][Ala³¹]IGF-1, and combinationsthereof. In a further embodiment, the IFG-1 analog is LongR3-IGF1, whichis a recombinant analog of human insulin growth factor-1. It iscontemplated that LongR3-IGF1 is initially present at a concentration ofapproximately 1 ng/ml to approximately 1000 ng/ml, more preferablyapproximately 5 ng/ml to approximately 500 ng/ml, more preferablyapproximately 50 ng/ml to approximately 500 ng/ml, more preferablyapproximately 100 ng/ml to approximately 300 ng/ml, or at aconcentration of approximately 100 ng/ml.

In certain embodiments, the compositions and methods of the presentinvention comprise transforming growth factor beta (TGF-β) or a TGF-βfamily member or variants or functional fragments thereof. As usedherein, the term “member of the TGF-β family” or the like refers togrowth factors that are generally characterized by one of skill in theart as belonging to the TGF-β family, either due to homology with knownmembers of the TGF-β family, or due to similarity in function with knownmembers of the TGF-β family. In particular embodiments of the invention,if the member of the TGF-β family is present, the TGF-β family member ofvariant or functional fragment thereof activates SMAD 2 or 3. In certainembodiments, the member of the TGF-β family is selected from the groupconsisting of Nodal, Activin A, Activin B, TGF-β, bone morphogenicprotein-2 (BMP2) and bone morphogenic protein-4 (BMP4). In oneembodiment, the member of the TGF-β family is Activin A.

It is contemplated that if Nodal is present, it is initially present ata concentration of approximately 0.1 ng/ml to approximately 2000 ng/ml,more preferably approximately 1 ng/ml to approximately 1000 ng/ml, morepreferably approximately 10 ng/ml to approximately 750 ng/ml, or morepreferably approximately 25 ng/ml to approximately 500 ng/ml. It iscontemplated that if used, Activin A is initially present at aconcentration of approximately 0.01 ng/ml to approximately 1000 ng/ml,more preferably approximately 0.1 ng/ml to approximately 100 ng/ml, morepreferably approximately 0.1 ng/ml to approximately 25 ng/ml, or mostpreferably at a concentration of approximately 10 ng/ml. It iscontemplated that if present, TGF-β is initially present at aconcentration of approximately 0.01 ng/ml to approximately 100 ng/ml,more preferably approximately 0.1 ng/ml to approximately 50 ng/ml, ormore preferably approximately 0.1 ng/ml to approximately 20 ng/ml.

In additional embodiments of the present invention, the compositions andmethods of the present invention are free of activators of FGFreceptors. There are currently at least 22 known members of the familyof fibroblast growth factors, with these factors binding to one of atleast one of four FGF receptors. As used herein, the term “activator ofan FGF receptor” refers to growth factors that are generallycharacterized by one of skill in the art as belonging to the FGF family,either due to homology with known members of the FGF family, or due tosimilarity in function with known members of the FGF family. In certainembodiments, the activator of an FGF receptor is an FGF, such as, butnot limited to α-FGF and FGF2. In particular embodiments, thecompositions and methods are free of exogenous FGF2. The phrase“exogenous FGF2” is used herein to indicate fibroblast growth factor 2,i.e., basic FGF, that is not intentionally added to the compositions ormethods of the present invention. Thus, in certain embodiments of thepresent invention, the methods and compositions are free ofintentionally supplied FGF2. The compositions or methods may, however,not necessarily be free of endogenous FGF2. As used herein, “endogenousFGF2” indicates that the cultured cells may be producing FGF2 of theirown accord when cultured according to the methods of the presentinvention. “Endogenous FGF2” also may be used to indicate residualimpurities from the primary cell culture or impurities from the startingmaterials. In specific examples, the compositions and methods of thepresent contain less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng/ml ofFGF2.

It is contemplated, however, that the compositions and methods of theinvention can include at least one activator of an FGF receptor,including any of the FGF polypeptides, functional fragments thereof orvariants thereof. It is contemplated that if FGF2 is present, it isinitially present at a concentration of approximately 0.1 ng/ml toapproximately 100 ng/ml, more preferably approximately 0.5 ng/ml toapproximately 50 ng/ml, more preferably approximately 1 ng/ml toapproximately 25 ng/ml, more preferably approximately 1 ng/ml toapproximately 12 ng/ml, or most preferably at a concentration ofapproximately 8 ng/ml. In another specific embodiment, the compositionsand methods of the invention can include at least one activator of anFGF receptor, other than FGF2. For example, the compositions and methodsof the present invention may comprise at least one of FGF-7, FGF-10 orFGF-22 or variants or functional fragments thereof. In specificembodiments, a combination of at least two of FGF-7, FGF-10 and FGF-22,or variants or functional fragments thereof, are present. In anotherembodiment, all three of FGF-7, FGF-10 and FGF-22, or variants orfunctional fragments thereof, are present. It is contemplated that ifany of FGF-7, FGF-10 or FGF-22 or variants or functional fragments arepresent, each is initially present at a concentration of approximately0.1 ng/ml to approximately 100 ng/ml, more specifically fromapproximately 0.5 ng/ml to approximately 50 ng/ml, more specificallyfrom approximately 1 ng/ml to approximately 25 ng/ml, more specificallyfrom approximately 1 ng/ml to approximately 12 ng/ml, or mostspecifically at a concentration of approximately 8 ng/ml.

In additional certain embodiments, the compositions and methods of thepresent invention comprise serum albumin (SA). In specific embodiments,the SA is either bovine SA (BSA) or human SA (HAS). In still morespecific embodiments, the concentration of the SA is more than about0.2%, volume to volume (v/v), but less than about 10% v/v. In even morespecific embodiments, the concentration of SA is more than about 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%,2.2%, 2.4%, 2.6%, 2.8%, 3.0%, 3.2%, 3.4%, 3.6%, 3.8%, 4.0%, 4.2%, 4.4%,4.6%, 4.8%, 5.0%, 5.2%, 5.4%, 5.6%, 5.8%, 6.0%, 6.2%, 6.4%, 6.6%, 6.8%,7.0%, 7.2%, 7.4%, 7.6%, 7.8%, 8.0%, 8.2%, 8.4%, 8.6%, 8.8%, 9.0%, 9.2%,9.4%, 9.6% and 9.8% (v/v).

In additional embodiments, the compositions and methods comprise atleast one insoluble substrate. For example, the differentiable cells maybe placed on a cell culture surface that comprises such compounds as,but is not limited to, polystyrene, polypropylene. The surface may, inturn, be coated with an insoluble substrate. In specific embodiments,the insoluble substrate is selected from the group consisting of acollagen, a fibronectin and fragments or variants thereof. Otherexamples of insoluble substrates include, but are not limited to,fibrin, elastin, fibronectins, laminins and nidogens.

Accordingly, the cell culture environments and methods of the presentinvention comprise plating the cells in an adherent culture. As usedherein, the terms “plated” and “plating” refer to any process thatallows a cell to be grown in adherent culture. As used herein, the term“adherent culture” refers to a cell culture system whereby cells arecultured on a solid surface, which may in turn be coated with aninsoluble substrate that may in turn be coated with another surface coatof a substrate, such as those listed below, or any other chemical orbiological material that allows the cells to proliferate or bestabilized in culture. The cells may or may not tightly adhere to thesolid surface or to the substrate. The substrate for the adherentculture may comprise any one or combination of polyornithine, laminin,poly-lysine, purified collagen, gelatin, fibronectin, tenascin,vitronectin, entactin, heparin sulfate proteoglycans, poly glycolyticacid (PGA), poly lactic acid (PLA), and poly lactic-glycolic acid(PLGA). Furthermore, the substrate for the adherent culture may comprisethe matrix laid down by a feeder layer, or laid down by the pluripotenthuman cell or cell culture. As used herein, the term “extracellularmatrix” encompasses solid substrates such as but not limited to thosedescribed above, as well as the matrix laid down by a feeder cell layeror by the pluripotent human cell or cell culture. In one embodiment, thecells are plated on MATRIGEL™-coated plates. In another embodiment, thecells are plated on fibronectin-coated plates. In certain embodiments,if the cells are plated on fibronectin, the plates are prepared bycoating with 10 μg/ml human plasma fibronectin (Invitrogen, #33016-015),diluted in tissue grade water, for 2-3 hours at room temperature. Inanother embodiment, serum can be placed in the medium for up to 24 hoursto allow cells to plate to the plastic. If using serum to promote theattachment of the cells, the media is then removed and the compositions,which are essentially serum-free, are added to the plated cells.

The compositions and methods of the present invention contemplate thatthe differentiable cells are cultured in conditions that are essentiallyfree of a feeder cell or feeder layer. As used herein, a “feeder cell”is a cell that grows in vitro, that is co-cultured with a target celland stabilizes the target cell in its current state of differentiation.As used herein, a “feeder cell layer” can be used interchangeably withthe term “feeder cell.” As used herein, the term “essentially free of afeeder cell” refers to tissue culture conditions that do not containfeeder cells, or that contain a de minimus number of feeder cells. By“de minimus”, it is meant that number of feeder cells that are carriedover to the instant culture conditions from previous culture conditionswhere the differentiable cells may have been cultured on feeder cells.In one embodiment of the above method, conditioned medium is obtainedfrom a feeder cell that stabilizes the target cell in its current stateof differentiation. In another embodiment, the defined medium is anon-conditioned medium, which is a medium that is not obtained from afeeder cell.

As used herein, the term “stabilize,” when used in reference to thedifferentiation state of a cell or culture of cells, indicates that thecells will continue to proliferate over multiple passages in culture,and preferably indefinitely in culture, where most, if not all, of thecells in the culture are of the same differentiation state. In addition,when the stabilized cells divide, the division typically yield cells ofthe same cell type or yield cells of the same differentiation state. Astabilized cell or cell population in general, does not furtherdifferentiate or de-differentiate if the cell culture conditions are notaltered, and the cells continue to be passaged and are not overgrown. Inone embodiment, the cell that is stabilized is capable of proliferationin the stable state indefinitely, or for at least more than 2 passages.In a more specific embodiment, the cells are stable for more than 3passages, 4 passages, 5 passages, 6 passages, 7 passages, 8 passages, 9passages, more than 10 passages, more than 15 passages, more than 20passages, more than 25 passages, or more than 30 passages. In oneembodiment, the cell is stable for greater than approximately 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, or 11 months of continuous passaging. In anotherembodiment, the cell is stable for greater than approximately 1 year ofcontinuous passaging. In one embodiment, stem cells are maintained inculture in a pluripotent state by routine passage in the defined mediumuntil it is desired that they be differentiated. As used herein, theterm “proliferate” refers to an increase in the number cells in a cellculture.

In certain embodiments, the compositions and methods comprise aninactivator of BMP signaling. As used herein, an “inactivator of BMPsignaling” refers to an agent that antagonizes the activity of one ormore BMP proteins or any of their upstream or downstream signalingcomponents through any of its possible signaling pathways. Thecompound(s) used to inactivate BMP signaling can be any compound knownin the art, or later discovered. Non-limiting examples of inactivatorsof BMP signaling include dominant-negative, truncated BMP receptor,soluble BMP receptors, BMP receptor-Fc chimeras, noggin, follistatin,chordin, gremlin, cerberus/DAN family proteins, ventropin, high doseactivin, and amnionless.

In certain embodiments, the compositions and methods can comprise atleast one hormone, cytokine, adipokine, growth hormone or variant orfunctional fragment thereof. It is currently contemplated that incertain embodiments, the growth hormone present in the defined mediumwill be of the same species as the differentiable cells that arecultured with the defined media. Thus, for example, if a human cell iscultured, the growth hormone is human growth hormone. The use of growthhormone that is from a species different than the cultured cells is alsocontemplated. Preferably the hormone, cytokine, adipokine and/or growthhormone is present at an initial concentration of approximately 0.001ng/ml to approximately 1000 ng/ml, more preferably approximately 0.001ng/ml to approximately 250 ng/ml, or more preferably approximately 0.01ng/ml to approximately 150 ng/ml.

Examples of cytokines and adipokines that may be included in thecompositions and methods of the present invention include, but are notlimited to, the four α-helix bundle family of cytokines, theinterleukin-1 (IL-1) family of cytokines, the IL-17 family of cytokinesand the chemokine family of cytokines. Of course, the inventioncontemplates members and subclasses of each of these families ofcytokines, such as, but not limited to, the CC chemokines, the CXCchemokines, the C chemokines and the CX₃C chemokines, interferons,interleukins, lymphotoxins, c-kit ligand, granulocyte-macrophagecolony-stimulating factor (GM-CSF), monocyte-macrophagecolony-stimulating factor (M-CSF), granulocyte colony-stimulating factor(G-CSF), leptin, adiponectin, resistin, plasminogen activatorinhibitor-1 (PAI-1), tumor necrosis factor-alpha (TNFα), tumor necrosisfactor-beta (TNFβ), leukemia inhibitory factor, visfatin, retinolbinding protein 4 (RBP4), erythropoietin (EPO), thrombopoietin (THPO).Of course, one of skill in the art will understand that the inventioncontemplates variants or functional fragments of the above-listedfactors.

The present invention relates to methods of culturing differentiablecells, with the methods comprising plating differentiable cells on acell culture surface, providing a basal salt nutrient solution to thecells and providing a means for stimulating ErbB2-directed tyrosinekinase activity in the cells.

In one embodiment, differentiable cells are contacted with the at leastone of the compositions of the invention in the absence of serum orserum replacement, and in the absence of a feeder cell layer, such thatthe cells are maintained in an undifferentiated state for at least onemonth. Pluripotency can be determined through characterization of thecells with respect to surface markers, transcriptional markers,karyotype, and ability to differentiate to cells of the three germlayers. These characteristics are well known to those of ordinary skillin the art.

In another embodiment, the differentiable cells are cultured insuspension, using the cell media of the present invention. The term“suspension” as used in the context of cell culturing is used as it isin the art. Namely, cell culture suspensions are cell cultureenvironments where the cells do not adhere to a surface. One of skill inthe art will be familiar with suspension culture techniques, including,but not limited to, the use of equipment such as flow hoods, incubatorsand/or equipment used to keep the cells in constant motion, e.g.,rotator platforms, shakers, etc, if necessary. As used herein, cells are“in motion” if they are moving, or if their immediate environment ismoving relative to the cells. If the cells are kept “in motion”, themotion will, in one embodiment, be a “gentle motion” that is designed toavoid or prevent exposing the cells to sheer stress.

In one particular embodiment, the differentiable cells are expanded in asuspension culture, using the cell media of the present invention. Inanother particular embodiment, the differentiable cells expand insuspension, but do not differentiate. The term “expand” in the contextof cell culture is used as it is the art, Namely, expand is used toindicate cellular proliferation to increase the number of viable cells.In a specific embodiment, the cells are expanded in a culture suspensionby culturing for more than about one day, i.e., about 24 hours. In amore specific embodiment, the cells are expanded in a suspension cultureby culturing for at least 2, 3, 4, 5, 6, 7 days, or at least 2, 3, 4, 5,6, 7, 8 weeks.

In general, the cell medium compositions of the present invention arerefreshed at least once every day, but the medium can be changed moreoften or less often, depending of the specific needs and circumstancesof the suspension culture. For example, the medium may be refreshedabout every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23 or 24 hours, or any fraction thereof. Inadditional examples, the medium may be refreshed less often such as, butnot limited to, every 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 orevery 2 or more days, or any time frame in between.

In general, the cells that are cultured in suspension in the mediumcompositions of the present invention are “split” every week or so, butthe cells can be split more often or less often, depending of thespecific needs and circumstances of the suspension culture. For example,the cells may be split every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14 or more days, or any time frame in between. As used herein, the term“split” in the context of cell culture is used as it is in the art.Namely, cell culture splitting, or passaging, is the collection of cellsfrom a previous culture and subsequent transfer of small number ofcollected (harvested) cells into a new cell culture vessel. In general,passaging cells allows the cells to continue to grow in a healthy cellculture environment. One of skill in the art will be familiar with theprocess and methods of cell culture passaging, which may, but notnecessarily, involve the use enzymatic or non-enzymatic methods that maybe used to disaggregate cells that have clumped together during theirgrowth expansion.

In some instances, a degree of cell death may occur in the cultured(suspended and adherent) cells immediately after passaging. In oneembodiment, the differentiable cells can “recover” from passaging, bydelaying the refreshing of the cell medium for more than 24 hours.Thereafter, the cell medium may be changed more frequently. In anotherembodiment, the cell culture medium can further comprise an inhibitor ofcell death. For example, Wartanabe et al., recently disclosed the use ofa Rho-associated kinase inhibitor, Y27632, to protect human ES cellsafter dissociation. See Watanbe, K., et al., Nat. Biotechnol.,25(6):681-686 (2007), which is incorporated by reference. In additionalembodiments, the cell culture medium may comprise caspase inhibitors,growth factors or other trophic factors to prevent or attenuate celldeath immediately after passaging. Specific examples of compounds thatmay be used include, but are not limited to, HA 1077, Dihydrochloride,Hydroxyfasudil, Rho Kinase Inhibitor, Rho-Kinase Inhibitor II, RhoKinase Inhibitor III, Kinase Inhibitor IV and Y27632 all of which arecommercially available. In still another embodiment, the compounds orfactors used to prevent or attenuate cell death during or immediatelyafter cell passaging may be removed from the cell culture medium afterthe cells have recovered from the passaging process.

In additional embodiments, the compositions and methods the presentinvention may also comprise the presence or use of surfactants. In oneparticular embodiment, the compositions and methods comprise at leastone surfactant in the context of a suspension culture. Surfactants arewell-known in the art and, generally speaking, are amphiphilic innature. In specific embodiments, the present invention comprises the useof at least one surfactant that is either anionic, cationic, non-ionicor zwitterionic. The concentration of the surfactant used in thecompositions and methods of the present invention is a matter of routinescreening and optimization. For example, Owen et al., reported the useof surfactants in cell culture techniques for HeLa cells and humanamniotic cells. See Owen et al., J. Cell. Sci., 32:363-376 (1978), whichis incorporated by reference. Examples of surfactants that may be usedinclude, but are not limited to, Sodium dodecyl sulfate (SDS), ammoniumlauryl sulfate, and other alkyl sulfate salts, Sodium laureth sulfate(SLES), Alkyl benzene sulfonate, Soaps, or fatty acid salts, Cetyltrimethylammonium bromide (CTAB) (hexadecyl trimethyl ammonium bromide),and other alkyltrimethylammonium salts, Cetylpyridinium chloride (CPC),Polyethoxylated tallow amine (POEA), Benzalkonium chloride (BAC),Benzethonium chloride (BZT), Dodecyl betaine, Dodecyl dimethylamineoxide, Cocamidopropyl betaine, Coco ampho glycinate, Alkyl poly(ethyleneoxide), Copolymers of poly(ethylene oxide) and poly(propylene oxide)such as Pluronic F68, Alkyl polyglucosides, such as, but not limited to,Octyl glucoside, Decyl maltoside, Fatty alcohols, Cetyl alcohol, Oleylalcohol, Cocamide MEA, cocamide DEA and cocamide TEA.

It is contemplated that the differentiable cells can be passaged usingenzymatic, non-enzymatic, or manual dissociation methods prior to and/orafter contact with the defined medium of the invention. Non-limitingexamples of enzymatic dissociation methods include the use of proteasessuch as trypsin, collagenase, dispase, and ACCUTASE™. In one embodiment,ACCUTASE™ is used to passage the contacted cells. When enzymaticpassaging methods are used, the resultant culture can comprise a mixtureof singlets, doublets, triplets, and clumps of cells that vary in sizedepending on the enzyme used. A non-limiting example of a non-enzymaticdissociation method is a cell dispersal buffer. Manual passagingtechniques have been well described in the art, such as in Schulz etal., 2004 Stem Cells, 22(7):1218-38. The choice of passaging method isinfluenced by the choice of extracellular matrix, if one is present, andis easily determined by one of ordinary skill in the art.

In one specific embodiment, methods of culturing differentiable cellscomprise providing a dissociation solution to a layer of differentiablecells that are contained in a culture chamber prior to dissociation,where the dissociation breaks apart the layer of cells into singlecells. After dissociation, the single cells are placed into a new tissueculture chamber with a stem cell culture solution, wherein the stem cellculture solution comprises a basal salt nutrient solution and an ErbB3ligand. Once cultured, the single stem cells are placed in conditionsthat permit growth and division of the single cells. In another specificembodiment, the methods of culturing differentiable cells compriseproviding a dissociation solution to an aggregation differentiable cellsthat are contained in a culture chamber prior, where the dissociationbreaks apart the aggregates of cells into single cells or smalleraggregates of cells.

The disaggregation solution used in the methods of the present inventioncan be any disaggregation solution capable of breaking apart ordisaggregating the cells into single cells, without causing extensivetoxicity to the cells. Examples of disaggregation solutions include, butare not limited to, trypsin, ACCUTASE™, 0.25% Trypsin/EDTA, TrypLE, orVERSENE™ (EDTA) and trypsin. The methods of the present invention neednot result in every cell of the confluent layer or suspension beingdisaggregated into single cells, provided that at least a few singlecells are disaggregated and capable of being re-cultured.

Either at the beginning of culture, or after passaging, thedifferentiable cells can be seeded at any density, including a singlecell in a culture chamber. The cell density of the seeded cells may beadjusted depending on a variety of factors, including but not limited tothe use of adherent or suspension cultures, the specific recipe of thecell culture media used, the growth conditions and the contemplated useof the cultured cells. Examples of cell culture densities include, butare not limited to, 0.01×10⁵ cells/ml, 0.05×10⁵ cells/ml, 0.1×10⁵cells/ml, 0.5×10⁵ cells/ml, 1.0×10⁵ cells/ml, 1.2×10⁵ cells/ml, 1.4×10⁵cells/ml, 1.6×10⁵ cells/ml, 1.8×10⁵ cells/ml, 2.0×10⁵ cells/ml, 3.0×10⁵cells/ml, 4.0×10⁵ cells/ml, 5.0×10⁵ cells/ml, 6.0×10⁵ cells/ml, 7.0×10⁵cells/ml, 8.0×10⁵ cells/ml, 9.0×10⁵ cells/ml, or 10.0×10⁵ cells/ml, ormore or any value in between.

Differentiable cells may also be utilized to screen for molecules orfactors that influence their plasticity or other characteristics. Forexample, differentiable cells could be used to identify agents thatinduce apoptosis, differentiation or proliferation, as well as similareffects in differentiated lineages that have been generated from thedifferentiable cells.

Because the compositions and methods of the present invention allow forsingle cell passaging, differentiable cells have been successfullycultured in high-throughput settings, such as, but not limited to,96-well plates and 384-well plates. FIG. 16 shows the morphology andalkaline phosphatase staining of BG02 cells that were cultured inDC-HAIF in both a 96-well and 384-well plate, using the methodsdescribed herein. Briefly, hESCs cells that were split, using ACCUTASE™,and plated in 96-well and 384-well plates and cultured showed a similarplating efficiency as what is observed using other culture dishes. Inaddition, the cells formed colonies, and were expanded successfully over5 days in the smaller environments. These smaller cultures remainedmorphologically undifferentiated, and stained uniformly positive foralkaline phosphatase, a marker of undifferentiated cells. Furthermore,hESCs could also be grown in 96-well culture devices (not shown) thatprovide real-time measurements of impedance, which can be used tomeasure cell proliferation and viability using the RT-CES™ methods fromACEA Biosciences, Inc. (www.aceabio.com). Such an approach would enablea label-free identification and quantitation of subtle or immediateeffects on differentiable cells, as well as measurements ofproliferation, apoptosis and changes to morphology, in real time.

The compositions and methods of the invention may contain virtually anycombination of the components set out above or described elsewhereherein, provided the compositions and methods comprise a basal saltnutrient solution and a means for stimulating ErbB2 directed tyrosinekinase activity. As one skilled in the art would recognize, thecomponents of the compositions and methods of the invention will varyaccording to the protocol design. Accordingly, one embodiment of thepresent invention relates to culturing differentiable cells in 96-wellplates and/or 384-well plates. Indeed, using the methods andcompositions of the present invention, the cell culture chamber, i.e.,the culture dish, is no longer limited to specific dimensions. Thus, themethods of the present invention is not limited to specific culturechamber dimensions.

The compositions and methods described herein have several usefulfeatures. For example, the compositions and methods described herein areuseful for modeling the early stages of human development. Furthermore,the compositions and methods described herein can also serve fortherapeutic intervention in disease states, such as neurodegenerativedisorders, diabetes mellitus or renal failure, such as by thedevelopment of pure tissue or cell type.

The cell types that differentiate from differentiable cells have severaluses in various fields of research and development including but notlimited to drug discovery, drug development and testing, toxicology,production of cells for therapeutic purposes as well as basic scienceresearch. These cell types express molecules that are of interest in awide range of research fields. These include the molecules known to berequired for the functioning of the various cell types as described instandard reference texts. These molecules include, but are not limitedto, cytokines, growth factors, cytokine receptors, extracellular matrix,transcription factors, secreted polypeptides and other molecules, andgrowth factor receptors.

It is contemplated that the differentiable cells of the invention can bedifferentiated through contact with a cell differentiation environment.As used herein, the term “cell differentiation environment” refers to acell culture condition wherein the differentiable cells are induced todifferentiate, or are induced to become a human cell culture enriched indifferentiated cells. Preferably, the differentiated cell lineageinduced by the growth factor will be homogeneous in nature. The term“homogeneous,” refers to a population that contains more thanapproximately 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the desired cell lineage.

A cell differentiating medium or environment may be utilized topartially, terminally, or reversibly differentiate the differentiablecells of the present invention. In accordance with the invention themedium of the cell differentiation environment may contain a variety ofcomponents including, for example, KODMEM medium (Knockout Dulbecco'sModified Eagle's Medium), DMEM, Ham's F12 medium, FBS (fetal bovineserum), FGF2 (fibroblast growth factor 2), KSR or hLIF (human leukemiainhibitory factor). The cell differentiation environment can alsocontain supplements such as L-Glutamine, NEAA (non-essential aminoacids), P/S (penicillin/streptomycin), N2, B27 and β-mercaptoethanol(β-ME). It is contemplated that additional factors may be added to thecell differentiation environment, including, but not limited to,fibronectin, laminin, heparin, heparin sulfate, retinoic acid, membersof the epidermal growth factor family (EGFs), members of the fibroblastgrowth factor family (FGFs) including FGF2 and/or FGF8, members of theplatelet derived growth factor family (PDGFs), transforming growthfactor (TGF)/bone morphogenetic protein (BMP)/growth and differentiationfactor (GDF) factor family antagonists including but not limited tonoggin, follistatin, chordin, gremlin, cerberus/DAN family proteins,ventropin, high dose activin, and amnionless or variants or functionalfragments thereof TGF/BMP/GDF antagonists could also be added in theform of TGF/BMP/GDF receptor-Fc chimeras. Other factors that may beadded include molecules that can activate or inactivate signalingthrough Notch receptor family, including but not limited to proteins ofthe Delta-like and Jagged families as well as inhibitors of Notchprocessing or cleavage, or variants or functional fragments thereof.Other growth factors may include members of the insulin like growthfactor family (IGF), insulin, the wingless related (WNT) factor family,and the hedgehog factor family or variants or functional fragmentsthereof. Additional factors may be added to promote mesendodermstem/progenitor, endoderm stem/progenitor, mesoderm stem/progenitor, ordefinitive endoderm stem/progenitor proliferation and survival as wellas survival and differentiation of derivatives of these progenitors.

The compositions described herein are useful for the screening of testcompounds to determine whether a test compound modulates pluripotency,proliferation, and/or differentiation of differentiable cells.Pluripotency, proliferation and/or differentiation of differentiablecells can be readily ascertained by one of ordinary skill in the art.Non-limiting methods include examining cell morphology, the expressionof various markers, teratoma formation, cell counts and measurements ofimpedance.

The progression of the differentiable cells to the desired cell lineage,or its maintenance in an undifferentiated state can be monitored byquantitating expression of marker genes characteristic of the desiredcell lineage as well as the lack of expression of marker genescharacteristic of differentiable cell types. One method of quantitatinggene expression of such marker genes is through the use of quantitativePCR (Q-PCR). Methods of performing Q-PCR are well known in the art.Other methods that are known in the art can also be used to quantitatemarker gene expression. Marker gene expression can be detected by usingantibodies specific for the marker gene of interest.

In certain embodiments, the screening method encompasses methods ofidentifying a compound capable of modulating pluripotency, proliferationand/or differentiation of a differentiable cell, comprising (a)providing a differentiable cell; (b) culturing the cell in a compositioncomprising a basal salt nutrient solution and an ErbB3 ligand, whereinthe composition is essentially serum free; (c) contacting the cell witha test compound; and determining whether an increase or decrease inpluripotency, proliferation and/or differentiation occurs in the cellcontacted with the compound, said increase being an indication that thecompound modulates pluripotency, proliferation and/or differentiation.In certain embodiments, the ErbB3 ligand is HRG-β. In other embodiments,the ErbB3 ligand can be substituted with a test compound, to determinethe effects of the test compound. For example, the effects onpluripotency, proliferation and/or differentiation that occurs with thetest compound can be compared to the effects on pluripotency,proliferation and/or differentiation that occurs with the ErbB3 ligandto determine the effects of the test compound on the differentiablecells. It is contemplated that any of the compositions described hereincan be used in the screening methods of the present invention.

In yet another embodiment, the cells can be cultured in the absence ofan ErbB3 ligand (ErbB2-directed tyrosine kinase activity) to determinethe effects of the absence of an ErbB3 ligand (ErbB2-directed tyrosinekinase activity) on the cells.

Using the methods described herein, compositions comprising the desiredcell lineage that are substantially free of other cell types can beproduced. Alternatively, compositions comprising mixtures of thedifferentiable cells and the desired cell lineage can also be produced.

In some embodiments of the present invention, cells of the desired celllineage can be isolated by using an affinity tag that is specific forsuch cells. One example of an affinity tag specific for a target cell isan antibody that is specific to a marker polypeptide that is present onthe cell surface of the target cell but which is not substantiallypresent on other cell types that would be found in a cell cultureproduced by the methods described herein.

The present invention also relates to kits, wherein the kit comprises abasal salt nutrient solution and at least one compound capable ofstimulating ErbB2-directed tyrosine kinase activity. In one embodiment,the kits comprise at least one ErbB3 ligand, as described herein. Inanother embodiment, the kits comprise more than one ErbB3 ligand. Inanother embodiment, the kits comprise at least one of TGF-β or a TGF-βfamily member or a variant or functional fragment thereof as describedherein. In yet another embodiment, the kits comprise more than one ofTGF-β or a TGF-β family member or a variant or functional fragmentthereof. In still another embodiment, the kits comprise at least onefibroblast growth factor or variant or functional fragment thereof. Inanother embodiment, the kits comprise more than one fibroblast growthfactor or variant or functional fragment thereof. In a specificembodiment, the kits comprise at least one of FGF-7, FGF-10, FGF-22 orvariants or functional fragments thereof. In another embodiment, thekits comprise serum albumin. In still another embodiment, the kitscomprise serum and/or at least one insoluble substrate as describedherein and/or at least one disaggregation solution.

The kits of the invention may contain virtually any combination of thecomponents set out above or described elsewhere herein. As one skilledin the art would recognize, the components supplied with kits of theinvention will vary with the intended use for the kits. Thus, kits maybe designed to perform various functions set out in this application andthe components of such kits will vary accordingly.

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in their entirety in order to more fullydescribe the state of the art to which this invention pertains.

EXAMPLES

The human embryonic stem cell line BG01v (BresaGen, Inc., Athens, Ga.)was used in some of the experiments described herein. The BG01v hESCline is a karyotypically variant cell line, which exhibits stablekaryotype containing specific trisomies (karyotype: 49, XXY, +12, +17).Parent cultures were maintained as described previously (Schulz et al.,2003, BMC Neurosci., 4:27; Schulz et al., 2004, Stem Cells,22(7):1218-38; Rosler et al., 2004, Dev. Dynamics, 229:259-274; Brimbleet al., 2004 Stem Cells Dev., 13:585-596). Briefly, the cells were grownin dishes coated with MATRIGEL™ or fibronectin, in conditioned mediafrom mouse embryonic fibroblasts (MEFs) (MEF-CM) comprising DMEM:F12with 20% KSR, 8 ng/ml FGF2, 2 mM L-Glutamine, 1× non-essential aminoacids, 0.5 U/ml penicillin, 0.5 U/ml streptomycin, 0.1 mMβ-mercaptoethanol (Sigma, St. Louis, Mo., USA), with collagenasepassaging.

The defined culture (DC) media tested herein comprised DMEM/F12, 2 mMGlutamax, 1× non-essential amino acids, 0.5 U/ml penicillin, 0.5 U/mlstreptomycin, 10 μg/ml transferrin (all from Invitrogen, Carlsbad,Calif., USA) 0.1 mM β-mercaptoethanol (Sigma), 0.2% fatty acid-freeCohn's fraction V BSA (Serologicals), 1× Trace Element mixes A, B and C(Cellgro) and 50 μg/ml Ascorbic Acid (Sigma). Variable levels ofrecombinant growth factors were used, including FGF2 (Sigma),LongR3-IGF1 (JRH Biosciences), Heregulin-β EGF domain (HRGβ, Peprotech),TGFβ (R&D systems), nodal (R&D systems), LIF (R&D systems), EGF (R&Dsystems), TGFα (R&D systems), HRGα (R&D systems), BMP4 (R&D systems),and Activin A (R&D Systems). LongR3-IGF1 is a modified version of IGF1that has reduced affinity for IGF1 binding proteins, some of which areexpressed in hESCs. DC-HAIF is the defined culture media as above,containing 10 ng/ml HRG-β, 10 ng/ml Activin A, 200 ng/ml LR-IGF1 and 8ng/ml FGF2. DC-HAI is defined culture media as above containing 10 ng/mlHRG-β, 10 ng/ml Activin A, and 200 ng/ml LR-IGF1. In both DC-HAIF andDC-HAI, the LR-IGF1 component can, of course be replaced with IFG1.

MATRIGEL™ coated dishes were prepared by diluting Growth Factor ReducedBD MATRIGEL™ matrix (BD Biosciences, Franklin Lakes, N.J., USA) to afinal concentration range of about 1:30 to about 1:1000 in coldDMEM/F-12. In one embodiment, the concentration of MATRIGEL™ is about1:200. 1 ml/35 mm dish was used to coat dishes for 1-2 hours at roomtemperature or at least overnight at 4° C. Plates were stored up to oneweek at 4° C. MATRIGEL™ solution was removed immediately before use.

For the tested conditions, parent cultures were plated into 6-welldishes for comparison of multiple conditions. Cultures were typicallyplated directly into the test conditions. The cultures were assessedevery day and graded based on morphological criteria 4 to 5 days afterplating. The grading scale of 1 to 5 involved examining the wholeculture and assessing overall proportion of undifferentiated colonies,their relative size, and proportion of colonies or parts of coloniesexhibiting obvious differentiation. Grade 5 indicates “ideal” cultures,with large undifferentiated colonies and negligible differentiation.Grade 4 indicates a very good culture, but with some obviousdifferentiation. Grade 3 indicates an acceptable culture, but witharound half the colonies exhibiting obvious differentiation. Grade 2cultures are predominantly differentiated, with occasional putativeundifferentiated cells. Grade 1 cultures contain differentiated coloniesor the cultures did not adhere or did not survive. Cultures thatexhibited good expansion of undifferentiated cells were passaged toassess longer-term culture in these conditions.

Example 1 Expression of ErbB1-3, Nrg1 and ADAM19 in BG01v Cells

Real time RT-PCR was used to demonstrate expression of ErbB1-3,Neuregulin and ADAM-19 in BG01v cells (FIG. 1). BG01v cells cultured inDC media as described above, containing 100 ng/ml LongR3-IGF1 (LR-IGF1),8 ng/ml FGF2 and 1 ng/ml Activin A were harvested and RNA was preparedusing the RNeasy mini kit (Qiagen) according to the manufacturer'sinstructions. First strand cDNA was prepared using the iScript kit(Biorad) and real time PCR was carried out using a MJ Research Opticonthermal cycler.

TaqMan assays on demand (Applied Biosystems) for ADAM19 (Hs00224960_ml),EGFR (Hs00193306_ml), ErbB2 (Hs00170433_ml), ErbB3 (Hs00176538_ml), NRG1(Hs00247620_ml), OCT4 (Hs00742896_sl) and control GAPDH were used withTaqMan universal PCR (Applied Biosystems). The real time amplificationplots are shown in FIG. 1, demonstrating expression of these transcriptsin undifferentiated BG01v cells.

Example 2 Inhibition of ErbB2 Slows Proliferation of BG01v Cells

The EGF domain family of ligands bind to the ErbB family of receptortyrosine kinases. To examine the effect of known inhibitors of ErbBtyrosine kinases in hESCs, BG01v cells were plated in 6 well trays onMATRIGEL™ diluted at 1:1000, in defined culture medium (DC) containing100 ng/ml LongR3-IGF1, 8 ng/ml FGF2 and 1 ng/ml Activin A. On the nextday, DMSO (carrier control), 50 nM-20 μM AG1478 (an ErbB1 inhibitor), or100 nM-20 μM AG879 (an ErbB2 inhibitor) was added with fresh medium. Thecells were cultured for an additional 5 days, with daily media changes.The cultures were then fixed and stained for alkaline phosphataseactivity.

Subconfluent colonies of AP+BG01v cells observed (FIGS. 2A, and B) incontrol and AG1478 cultured cells, indicating that neither DMSO norAG1478 (50 nM-20 μM) had an apparent affect on cell proliferation.AG879, however, substantially inhibited cell growth at 5 μM (FIG. 2C)and caused cell death at 20 μM (not shown). The cultures grown in AG879did not appear to differentiate and appeared to maintain a pluripotentmorphology and alkaline phosphatase activity, indicating that AG879appeared to inhibit proliferation without inducing differentiation,suggesting that BG01v cells are reliant on ErbB2 signaling for cellsurvival. Conversely, BG01v cells grown in similar conditions as abovedo not appear to be reliant on ErbB 1 signal for proliferation.

Example 3 BG01v cells are Maintained in Defined Media ContainingHeregulin

Expression of ErbB2 and ErbB3 and the inhibition of proliferation withAG879 suggested that BG01v cells have active endogenous ErbB signalingand that they may also respond to exogenous HRG-β. BG01v cells weregrown in DC medium containing 10 ng/ml HRG-β, 200 ng/ml LongR3-IGF1, 8ng/ml FGF2 and 10 ng/ml Activin A, on MATRIGEL™ diluted 1:1000 (FIGS. 3Aand B). These cells were grown for 4 passages, or >20 days, exhibitedundifferentiated morphology and did not show elevated spontaneousdifferentiation.

Furthermore, BG01v cells were also maintained for 2 passages, or >13days, in DC medium comprising 10 ng/ml HRGβ, 200 ng/ml LongR3-IGF1, and10 ng/ml Activin A. These cultures proliferated normally and exhibitedvery low spontaneous differentiation, demonstrating that BG01v cellscould be maintained in defined conditions with HRGβ in the absence ofFGF2.

Example 4 The Role of ErbB2-Directed Tyrosine Kinase in ES Cells

RT-PCR demonstrated that mESCs express ADAM19, Neuregulin1 (Nrg1), andErbB1-4 (FIG. 4A). In mESCs, ErbB2 and 3 appeared to be expressed athigher levels than ErbB1, with low levels of ErbB4 being detected. Thesedata suggest that endogenous HRG-β could be involved in driving mESCself-renewal.

The expression of the ErbB receptor transcripts in mouse embryonicfibroblasts (MEFs) was also examined (FIG. 4B). MEFs are a heterogenouspopulation of cells derived from E12.5-13.5 viscera that have been usedhistorically to maintain mouse and human EC cells and ES cells.Expression of Nrg1 and Adam19 in this population suggests that the HRG-βectodomain is also present in MEF-conditioned media and may exertsignificant effects upon pluripotency.

AG1478 and AG879 were used to examine the role of HRG/ErbB signaling inmouse ES cells. R1 mouse ES cells were maintained in standard conditionsin DMEM, 10% FBS, 10% KSR, 0.5 U/ml penicillin, 0.5 U/ml streptomycin,1×NEAA, 1 mM sodium pyruvate, 1000 U/ml LIF (ESGRO), 0.1 mM β-ME, andwere passaged with 0.5% trypsin/EDTA. 2×10⁵ cells/well were plated in 6well trays on MATRIGEL™ diluted at 1:1000. The day after plating, DMSO(carrier control), 1-50 μM AG1478, or 1-50 μM AG879 was added with freshmedium. The cells were cultured an additional 8 days, with daily mediachanges. The cultures were then fixed and stained for alkalinephosphatase activity.

DMSO and 1-50 μM AG1478 had no apparent affect on cell proliferation,with subconfluent colonies of alkaline phosphatase positive mESCsobserved (FIGS. 5A-C). However, AG879 substantially inhibited cellgrowth at 50 μM (compare FIGS. 5D and 5F) and may have slowedproliferation at 20 μM (FIG. 5E). mESCs grown in AG879 did not appear todifferentiate and maintained a pluripotent morphology, and alkalinephosphatase activity.

The results indicate that AG879 appeared to inhibit proliferation,without inducing differentiation, of mESCs, suggesting that mESCsrequire ErbB2 signaling for proliferation. Conversely, mESCs do notappear to be reliant on an ErbB1 signal for proliferation. Theconcentration of AG879 required to inhibit proliferation was ˜10× higherfor mESCs than that for BG01v cells grown in defined conditions,indicating that either the serum used in the mESC conditions may haveinterfered with the activity of the drug, that AG879 has a loweraffinity for the mouse ErbB2 tyrosine kinase than for human ErbB2tyrosine kinase, or that ErbB2 may play slightly different roles withthe different species of ES cells.

Another highly selective inhibitor of the ErbB2 tyrosine kinase,tyrphostin AG825 (Murillo, et al. 2001 Cancer Res 61, 7408-7412), wasused to investigate the role of ErbB2 in human ESCs. AG825 significantlyinhibited proliferation of hESCs growing in conditioned medium (CM)(FIG. 6A). AG825 inhibited proliferation without widespread cell death,and viable hESCs could be maintained for >5 days (not shown). Westernblotting showed that AG825 inhibited autophosphorylation of ErbB2 attyrosine-1248 in starved/heregulin (HRG) pulsed hESCs growing in DC-HAIF(FIG. 6B). Thus, disruption of ErbB2 signaling severely inhibited hESCproliferation. To establish hESCs in defined growth conditions, culturescould be passaged directly from CM conditions into DC-HAIF and exhibitedminimal spontaneous differentiation (FIG. 6C). Colony and cell-countingassays confirmed that LongR3-IGF1 and HRG played the major roles inself-renewal and proliferation in the context of one of the embodimentsof the present invention (FIG. 6D, 6E). Phosphorylation of IGF1R, IR,FGF2α, ErbB2, and ErbB3 was also observed in both steady-state DC-HAIFcultures, and in starved cultures that were pulsed with DC-HAIF (FIG.6F).

Example 5 Culture of Mouse ES Cells in Defined Conditions

To further examine the role of HRG/ErbB2 signaling in mouse ES cells,the proliferation of R1 ES cells was examined in DC medium using acombination of growth factors. 1×10⁵ cells/well were plated in 6-welltrays, coated with 0.2% gelatin, in DC containing combinations of 10ng/ml HRG-β, 100 ng/ml LongR3-IGF1, 1 ng/ml Activin A, 1000 U/ml mouseLIF or 10 ng/ml BMP4 (Table 1, below). Proliferation was observed over 8days.

Viable colonies only grew in conditions containing at least LIF/HRG-β orLIF/BMP4 (Table 1). No additional obvious benefit was observed whenLongR3-IGF1 or Activin were added to these combinations. Normalproliferation was observed in a control parental culture, and no viablecolonies were observed in defined media without any growth factors.

TABLE 1 HRG IGF Activin LIF BMP4 Growth + No + + Yes + + No + + +Yes + + + No + + + + Yes + + No + + + Yes + + Yes + + + Yes

A quantitative assay was performed by plating 2×10⁵ cells/well in 6-welltrays on 1:1000 MATRIGEL™, in selected combinations of 10 or 50 ng/mlHRG-β, 10 ng/ml EGF, 1000 U/ml LIF or 10 ng/ml BMP4. The cultures weregrown for 8 days, fixed, and the number of alkaline phosphatase colonieswas counted (FIG. 7A). No colonies were observed in defined conditionswithout growth factors, and <45 colonies were observed with HRG-β,HRG-β/EGF and HRG-β/BMP combinations. While 1358 colonies were observedin LIF alone, 4114 and 3734 colonies were observed in the 10 ng/mlHRG-β/LIF and 50 ng/ml HRG-β/LIF combinations, respectively. Thisindicated that in defined conditions, LIF alone provided a substantialpluripotency signal, and HRG-β exhibited a large synergistic effect withLIF, more than doubling the number of proliferating mESC colonies inthis assay. Low magnification images of this assay also indicate thissynergistic proliferative effect (FIGS. 7B-G).

Example 6 Characterization of Pluripotency of Human Embryonic Stem Cells(hESCs) Maintained in DC-HAIF

Multiple approaches were used to confirm the maintenance of plasticityof hESCs in DC-HAIF. BG02 cells cultured in DC-HAIF for 6 months (25passages) maintained the potential to form complex teratomas (FIG. 8A)and representatives of the three germ layers in vitro (FIG. 8B).Transcriptional analyses were used to compare global expression in hESCscells (Liu et al 2006, BMC Dev Biol 6, 20) maintained in CM and DC-HAIF.Greater than 11,600 transcripts were detected in BG02 cells grown inDC-HAIF for 10 and 32 passages, and BG02 cells grown in CM for 64passages. There were about 10364 transcripts common to all populations(FIG. 8C), including known hESC markers such as CD9, DNMT3, NANOG, OCT4,TERT and UTF1 (not shown). High correlation coefficients were observedin comparisons of CM and DC-HAIF cultures (R²select=0.928), as well asin early and late passage cells (R²select=0.959) (FIG. 8D). Hierarchicalclustering analysis demonstrated that BG02 cells maintained in DC-HAIFgrouped tightly and retained a close similarity to BG02 and BG03 cellsmaintained in CM (FIG. 8E). These data are consistent with previousanalyses showing that undifferentiated hESCs clustered tightly comparedto embryoid bodies or fibroblasts (Liu et al 2006, BMC Dev Biol 6, 20).Thus, cells maintained in the compositions of the present invention areable to maintain key markers of pluirpotency. Accordingly, thecompositions of the present invention can be used as a simple medium forsupporting self-renewal of differentiable cells.

Example 7 Maintenance of Human Embryonic Stem Cells (ESCs) on HumanizedExtracellular Matrices (ECMs) in DC-HAIF

To investigate the role of ErbB2 signaling and develop a defined mediafor hESCs, DC-HAIF cultures were initially expanded on culture dishedcoated with growth factor-reduced MATRIGEL™ 1:30, but could also bemaintained successfully long-term on this substrate diluted 1:200 (FIG.9A), or 1:1000. BG02 and CyT49 hESCs could also be maintained for >5passages on tissue culture dishes coated with human serum (FIG. 9B);human fibronectin (FIG. 9C); or VITROGRO™ (FIG. 9D), which is aproprietary humanized ECM.

Example 8 Single Cell Passaging of Human Embryonic Stem Cells (ESCs)

Multiple research groups have demonstrated that certain triplodies,notably of hChr12 and 17, are accumulated in hESCs under certainsub-optimal culture conditions (Baker et al., 2007 Nat.Biotech.25(2):207-215). The appearance of triploidies seems to be mostdirectly related to poor cell survival when cultures are split to singlecells at passaging, providing a presumed strong selective growthadvantage for cells harboring these aneuploidies. Conversely, hESCsgrowing in one embodiment of the present invention, DC-HAIF, maintainedhigh viability at plating after being split to single cells (FIG.10A-D). BG01 and BG02 cells maintained a normal karyotype (FIG. 10E)after being passaged with ACCUTASE™ for >18 and 19 passagesrespectively. The maintenance of normal karyotype in cells demonstratedthat disaggregation of hESC cultures to single cells did not inherentlylead to the accumulation of these trisomies in hESCs maintained inDC-HAIF. BG01 and BG02 cultures were also passaged by disaggregation tosingle cells with multiple passaging agents (FIG. 11). Cultures weresplit with ACCUTASE™, 0.25% Trypsin/EDTA, TrypLE, or VERSENE™ (EDTA) for5 passages and karyotyped. The data demonstrate that culturing andpassaging hESCs in the compositions of the present invention maintaineda normal karyotype in at least two human embryonic cell lines, using avariety of cell disaggregation reagents.

Large-scale expansion of undifferentiated hESCs is also possible, usingthe compositions of the present invention. A starting confluent cultureof BG02 cells in a 60 mm plate was expanded in DC-HAIF through 4passages to generate >1.12×10¹⁰ cells in 20 days in a single experiment.The cultures remained undifferentiated, as demonstrated by >85% of thecells in the batch maintaining expression of markers of pluirpotencysuch as OCT4, CD9, SSEA-4, TRA-1-81 when examined by flow cytometry(FIG. 12A). Expression of other markers of pluripotency was alsoobserved by RT-PCR analysis, while markers of differentiated lineagesα-fetoprotein, MSX1 and HAND1 were not detected (FIG. 12B). Fluorescencein situ hybridization analysis demonstrated that the cells cultured andpassaged in DC-HAIF maintained expected copy numbers for hChr12 (98%2-copy), hChr17 (98% 2-copy), hChrX (95% 1-copy) and hChrY (98% 1-copy)(FIG. 12C). Karyotyping analysis also demonstrated that a normal euploidchromosome content and banding profile was maintained in these cells

Example 9 Insulin and IGF1 Exert Different Effects on hESCs when Appliedat Physiological Concentrations

Essentially all of the reported culture conditions for hESCs to dateinclude supraphysiological levels of insulin, which can stimulate bothIR and IGF1R. To distinguish the activities that insulin andinsulin-substitutes exert, compared to IGF1, hESCs are cultured indefined media conditions in physiological levels of these growthfactors. The concentrations of insulin and IGF1 are titrated from about0.2 to about 200 ng/ml and cell proliferation is monitored by countingcells after 5 days. Cultures that expand successfully are seriallypassaged 5 times. Physiological levels of IGF1 support the expansion ofhESC cultures, whereas physiological levels of insulin do not,indicating that the activity of insulin or insulin-substitutes cannotreplace IGF1, and that IGF1 and insulin (or insulin substitutes)represent separate classes of biological activities with regard toaction on hESCs.

Example 10 Methods for Screening the Effects of Supplements

To initially examine the effects of Vitamin B₁₂ and Vitamin B₆ on thegrowth or differentiation hESCs growing at an intermediate density, BG02cells are split using ACCUTASE™ and 1×10⁵ cells/well are plated in6-well trays in defined culture (DC) media. The DC media contains 10ng/ml HRG-β, 200 ng/ml LongR3-IGF1, and 10 ng/ml FGF10. Vitamin B₆ (0.5μM) and/or Vitamin B₁₂ (0.5 μM) are added to experimental wells. Cellnumbers in each condition are counted after 7 days. Cell counting andcolony counting of both experimental and control wells will provideinsight on the effects of Vitamin B₆ and Vitamin B₁₂ on cell growth.

In addition, markers of differentiation, such as OCT4 can be assayed inthe experimental well to determine the effects of the additives andsupplements to the differentiation state of the differentiable cells.

Example 11 Culturing of hESCs in the Absence of FGF2

BG02 cells were maintained long term in DC-HAI, for 20 passages (FIG.13A), and BG01 cells were also serially passaged in DC-HAI, both in theabsence of FGF2. The cultures did not deteriorate or exhibit overtdifferentiation, and exhibited normal expansion of undifferentiatedcolonies throughout the culture period. The maintenance of a normal malekaryotype in a BG02 culture was demonstrated after 6 passages in DC-HAI(FIG. 13B, 20/20 normal metaphase spreads).

Transcriptional analyses were used to compare global expression in hESCscells maintained in DC-HAIF and DC-HAI. Total cellular RNA was isolatedfrom hESCs using Trizol (Invitrogen) and was treated with DNase I(Invitrogen) according to the manufacturer's suggested protocol. Sampleamplification was performed with 100 ng of total RNA using the IlluminaRNA Amplification kit and labeling was achieved by incorporation ofbiotin-16-UTP (Perkin Elmer Life and Analytical Sciences) at a ratio of1:1 with unlabeled UTP. Labeled, amplified material (700 ng per array)was hybridized to Illumina Sentrix Human-6 Expression Beadchipscontaining 47,296 transcript probes according to the manufacturer'sinstructions (Illumina, Inc.). Arrays were scanned with an Illumina BeadArray Reader confocal scanner and primary data processing, backgroundsubtraction, and data analysis were performed using Illumina BeadStudiosoftware according to the manufacturer's instructions. A minimumdetection confidence score of 0.99 (a computed cutoff indicating thetarget sequence signal was distinguishable from the negative controls)was used to discriminate the presence or absence of transcriptexpression. Data analysis was performed using parallel approachesdescribed for other hESC samples (Liu et al 2006, BMC Dev Biol 6:20).Hierarchical clustering was performed as described previously (Liu et al2005, BMC Dev Biol 6:20), and was based on average linkage and Euclideandistances as the similarity metric using differentially expressed genesidentified by ANOVA (p<0.05). Detailed descriptions of the sensitivityand quality control tests used in array manufacture and algorithms usedin the Bead studio software are available from Illumina, Inc (San Diego,Calif.). The majority of transcripts detected were expressed in bothDC-HAIF and DC-HAI BG02 cultures, including known hESC markers such asCD9, DNMT3, NANOG, OCT4, TERT and UTF1 (not shown). High correlationcoefficients were observed in comparisons of DC-HAIF and DC-HAI cultures(R² select=0.961) (FIG. 14). Hierarchical clustering analysisdemonstrated that BG02 cells maintained in DC-HAI grouped tightly andretained a close similarity to cells maintained in DC-HAIF, as well asBG02 and other hESC lines in multiple culture formats (FIG. 15). Thesedata are consistent with previous analyses showing that undifferentiatedhESCs clustered tightly compared to embryoid bodies or fibroblasts (Liuet al 2006, BMC Dev Biol 6:20).

Furthermore, BG02 cells maintained in DC-HAI differentiated torepresentatives of mesoderm, endoderm and ectoderm in complex teratomasformed in SCID-beige mice (not shown), formally demonstrating themaintenance of pluripotency in cultures grown in the absence ofexogenous FGF2.

To examine if exogenous FGF2 was required in the context of single cellpassaging, BG01 cells were passaged with ACCUTASE™ and grown in definedconditions containing only 10 ng/ml HRG-β and 200 ng/ml LongR3-IGF1(DC-HI). These DC-HI cultures were maintained for 10 passages, and didnot exhibit overt differentiation or a slowing of proliferation.

These studies clearly demonstrated that the provision of exogenous FGF2is not required when hESCs are maintained in defined media minimallycontaining heregulin and IGF1. Furthermore cultures absent FGF2 retainedkey properties of pluripotency, including transcriptional profile anddifferentiation to mesoderm, endoderm and ectoderm in vivo.

Example 12 Suspension Cultures

Starting cultures of BG02 cells were maintained in DC-HAIF medium ondishes coated with 1:200 matrigel, as described herein and were split bypassaging with ACCUTASE™. To initiate suspension culture, BG02 cellswere disaggregated with ACCUTASE™ and placed in low attachment 6-welltrays at a density of 1.6, 3, or 6×10⁵ cells/ml (0.5, 1, or 2×10⁶ cellsin 3 ml volumes) in DC-HAIF medium. The trays were placed on a rotatingplatform at 80-100 rpm in a humidified incubator with 5% CO₂. Underthese conditions hESCs coalesced into small spheres of morphologicallyviable cells within 24 hours.

The medium in the wells was changed on the second day, and every daythereafter. Suspension aggregates continued to proliferate, growinglarger over time without obvious signs of differentiation (FIG. 17).Some of the spheres continued to aggregate over the course of theculture, as some aggregates became much larger than the majority. Inaddition, non-spherical aggregates could be observed in the process ofmerging during the first few days of the culture. To limit thiscontinued aggregation, 38 μg/ml DNaseI was included in some suspensioncultures for the first 24 hours. This approach appeared to conducive tothe initial aggregation, with relatively larger, but fewer, aggregatesformed in the presence of DNaseI. It is not clear, however, if theDNaseI treatment reduced the subsequent merging of spheres and exposureto DNaseI consistently made these aggregates harder to break up whensplitting.

Suspension cultures were disaggregated with ACCUTASE™ approximatelyevery 7 days and new spheres were established. While the densitiesvaried in different experiments, spheres established within this rangeof densities (1.6-6×10⁵ cells/ml) could be maintained in culture formore than 12 passages, or >80 days, without morphological signs ofdifferentiation. FISH analyses of serially passaged suspension hESCswere also performed to assess the chromosome number for commonaneuplodies. BG02 cells that had been grown in suspension for 6 passagesexhibited normal counts for hChr 12 (96% two copy, n=788), hChr 17 (97%two copy, n=587), hChr X (97% one copy, n=724) and hChr Y (98% one copy,n=689).

Example 13 Expansion of Differentiable Cells in Suspension Culture

Unlike embryoid body culture in the presence of serum or inducers ofdifferentiation, suspension aggregates of hESCs in DC-HAIF did notappear to differentiate. Obvious visceral endoderm was not observed,neither was the formation of structures resembling proamniotic cavities,both classic signs of embryoid body differentiation. To examine the lackof differentiation more closely, cultures were plated back into adherentconditions on MATRIGEL™ diluted 1:200 and cultured in DC-HAIF. Thesecultures were also primarily undifferentiated, and did not exhibitobvious morphological signs of increased differentiation such as thepresence of larger, flattened cells, or structured regions.

Cell counting was used to assess the relative growth rates of cells insuspension compared to adherent culture. In this experiment, an adherentculture of BG02 cells was passaged with ACCUTASE™, and about 1×10⁶ cellswere placed in parallel suspension or adherent culture wells. Individualwells were counted on days 1-6 and plotted on a log scale (FIG. 18).While a higher initial proportion of hESCs were viable after 24 hours inadherent culture (˜90% vs ˜14%), growth rates were comparablethereafter. This indicated that hESCs could proliferate just as rapidlyin suspension culture as in traditional adherent culture. Cell countsperformed during passaging allows one to gauge the amount of expansionpossible in this simple suspension system. In several cultures seededwith 5×10⁵ cells, approximately 10⁷ cells, or more, were generated after7 days. The expansion after 7 days in suspension culture equated toabout a 20-fold or more expansion, with the largest expansion observedbeing ˜24× the input cell number.

Example 14 Characteristics of Differentiable Cells Expanded inSuspension Culture

Quantitative RT-PCR (qPCR) was used to compare gene expression in hESCsgrown in suspension and adherent culture in DC-HAIF. Comparable levelsof OCT4, a marker of pluripotent cells, were observed in both cultureformats, confirming that cultures maintained in suspension wereprimarily undifferentiated. SOX17, a marker of definitive endoderm, wasnot expressed in either population of hESCs. The qPCR analysis alsoexamined the potential of suspension hESCs to differentiate todefinitive endoderm, as aggregates in suspension. Adherent andsuspension hESCs were differentiated using parallel conditions. hESCcultures were treated with RPMI containing 2% BSA, 100 ng/ml Activin A,8 ng/ml FGF2 and 25 ng/ml Wnt3A for 24 hours, followed by 2 days in thesame medium without Wnt3A. The expression of OCT4 was downregulated, andexpression of SOX17 upregulated similarly in both definitive endodermsamples compared to undifferentiated hESCs. This differentiationanalysis confirmed that hESCs cultured in suspension in DC-HAIFmaintained their differentiation potential, as evidenced by the likelyformation of definitive endoderm.

Example 15 Addition of an Apoptosis Inhibitor in Suspension Culture

To attenuate the loss of cells after initial passaging in suspension, aninhibitor of apoptosis was added to the medium. Cells were passaged asin Example 12, except that Y-27632, an inhibitor of p160-Rho-associatedcoiled-coil kinase (ROCK), was added to the medium.

Suspension aggregates of BG02 cells were formed by seeding 2×10⁶ singlecells in 6-well dishes in 3 ml DC-HAIF medium, at 100 rpm on a rotatingplatform in an incubator (Table 2, Experiment A). 10 μM Y27632 ROCKinhibitor was added to test wells for the course of the experiment andthe cultures observed daily and counted after 24 hours (day 1) and after4 or 5 days. As shown in FIG. 20, addition of Y27632 had a profoundeffect on the initial aggregation phase of suspension culture. Comparedto cells aggregated in medium without inhibitor, much larger aggregateswere formed in the presence of Y27632 (FIG. 20). Cell counting confirmedthat more viable cells were present in the presence of inhibitor (Table2, Experiment A). This difference in cell number persisted throughoutthe course of the culture period, with more cells also observed on day4, compared to cultures without inhibitor. As with previous suspensionculture experiments, cells exposed to Y27632 could also be seriallypassaged, and maintained in an undifferentiated state (not shown). Whenthe aggregates were split again, almost twice as many cells wereobserved with Y27632 treatment (Table 2 Experiment A). RT-PCR analysisdemonstrated that BG02 cells grown in suspension culture in the presenceof Y27632 remained undifferentiated (FIG. 21).

As previous experiments had shown that growth rates of cells insuspension and adherent culture were similar after the initial 24 hours,an experiment was performed where Y27632 was removed after this initialperiod (Table 2, Experiment B). Consistent with these previousobservations, Y27632 enhanced initial survival and aggregation of hESCsafter initial passage, but removing the inhibitor after 24 hours did notnegatively impact the number and viability count of cells analyzed onday 5. 1.4×10⁷ (+Y27632) and 1.8×10⁷ (+/−Y27632) viable cells weregenerated when inhibitor was present compared to 3.9×10⁶ cells inuntreated cultures. This analysis confirmed that Y27632 had the largestimpact during the first 24 hours of suspension hESC culture.

Because of the enhanced survival and aggregation observed in thepresence of Y27632, an experiment was performed to examine if it waspossible to reduce the number of cells used to seed suspension cultures(Table 2, Experiment C). Previous experiments had indicated that seedingES cells at a low density of about 5×10⁵ cells per 3 ml DC-HAIF, orless, did not work well. To determine if addition of a ROCK inhibitorwould allow cell seeding at lower densities, a range of cellconcentrations (from about 2×10⁶ total cells down to about 1×10⁵ totalcells was used to seed suspension cultures in 6-well trays cells in 3 mlDC-HAIF. 10 μM Y27632 was added to all conditions, and the cell numberand viability assessed on day 5. Successful aggregation and expansionwas observed even at low seeding densities. An approximate 13 foldexpansion of viable cells was observed even cultures that were onlyseeded with 1×10⁵ cells. Inhibition of ROCK with Y27632 thereforefacilitated initial survival of hESCs at much lower densities in thissuspension system.

TABLE 2 Suspension Cultures with and without an Apoptosis Inhibitor Cellcounts: total (viable, %) Expt. Treatment Seeding p0, day 1 p0, day 4p1, day 4 A HAIF 2 × 10⁶ 1.9 × 10⁶ (3.5 × 10⁵, 19%) 1.8 × 10⁶ (1.3 ×10⁶, 75%) 2.5 × 10⁶ (2.2 × 10⁶, 88%) +Y27632 2 × 10⁶ 1.6 × 10⁶ (1.2 ×10⁶, 74%) 7.8 × 10⁶ (7.1 × 10⁶, 91%) 4.6 × 10⁶ (4.2 × 10⁶, 91%) p0, day5 B HAIF 2 × 10⁶ 2.9 × 10⁶ (5.5 × 10⁵, 26%) 4.8 × 10⁶ (3.9 × 10⁶, 81.3%)+Y27632 2 × 10⁶ 1.9 × 10⁶ (1.4 × 10⁶, 73%) 1.5 × 10⁷ (1.4 × 10⁷, 92%)+/−Y27632 2 × 10⁶ N/A 1.9 × 10⁷ (1.8 × 10⁷, 96%) p0, day 5 C +Y27632 2 ×10⁶ 1.9 × 10⁶ (1.6 × 10⁶, 84%) 1.4 × 10⁷ (1.2 × 10⁷, 90%) 1 × 10⁶ 8.7 ×10⁵ (6.6 × 10⁵, 76%) 8.6 × 10⁶ (7.8 × 10⁶, 91%) 5 × 10⁵ 4.6 × 10⁵ (3.5 ×10⁵, 75%) 5.7 × 10⁶ (5.3 × 10⁶, 93%) 2.5 × 10⁵   2.6 × 10⁵ (2.3 × 10⁵,91%) 2.7 × 10⁶ (2.5 × 10⁶, 91%) 10⁵ 6.8 × 10⁴ (5.4 × 10⁴, 79%) 1.4 × 10⁶(1.3 × 10⁶, 92%) Expt. = Experiment; p0 = passage 0, p1 = passage 1; N/A= not available; Cell counts and percentages are rounded to 1 and 0decimal places, respectively.

Example 16 Suspension Cultures in Various Media

To determine if suspensions of ES cells could be cultured in the absenceof FGF2 and/or Activin A, ES cells were cultured in a variety of media,with and without these factors. Table 3 shows cell counting results fromsuspension cultures and indicters that suspension cultures could besuccessfully expanded in the absence of exogenous FGF2 (HAI conditions),as well as without exogenous FGF2 or Activin A (HI conditions). Theaddition of Y27632 increased the yield of cells generated by day 5 inall conditions. In addition, the cells in each media were successfullypassaged with no morphological signs of differentiation.

TABLE 3 Suspension Cultures in Various Media Cell counts: total (viable,%) Treatment Seeding p0, day 5 Fold Expansion HAIF 2 × 10⁶ 7.7 × 10⁶(6.5 × 10⁶, 83%) 3.25 HAI 2 × 10⁶ 7.0 × 10⁶ (6.3 × 10⁶, 91%) 3.15 HI 2 ×10⁶ 6.4 × 10⁶ (5.3 × 10⁶, 83%) 2.65 HAIF + Y27632 2 × 10⁶ 1.5 × 10⁷ (1.3× 10⁷, 90%) 6.5 HAI + Y27632 2 × 10⁶ 1.5 × 10⁷ (1.3 × 10⁷, 91%) 6.5 HI +Y27632 2 × 10⁶ 1.9 × 10⁷ (9.2 × 10⁶, 49%) 4.6

1. A method of expanding differentiable cells in suspension, said methodcomprising a) placing the differentiable cells in a suspension of cellmedium, the medium comprising a basal salt nutrient solution and anErbB3 ligand, where the cell medium is essentially free of serum; and b)maintaining the differentiable cells in the suspension of culture mediumand in appropriate culture conditions for more than one day, wherein thecell medium is changed periodically, wherein the differentiable cellsexpand and do not differentiate.
 2. The method of claim 1, wherein thecell medium is free of exogenous insulin and insulin substitutes.
 3. Themethod of claim 2, wherein the cell medium further comprisinginsulin-like growth factor or a functional fragment thereof.
 4. Themethod of claim 2, wherein the ErbB3 ligand in the cell medium isselected from the group consisting of Neuregulin-β, Heregulin-β (HRG-β),Heregulin-α (HRG-α), Neu differentiation factor (NDF), acetylcholinereceptor-inducing activity (ARIA), glial growth factor 2 (GGF2),motor-neuron derived factor (SMDF), Neuregulin-2, Neuregulin-2β(NRG2-β), Epiregulin, Biregulin and functional fragments thereof.
 5. Themethod of claim 4, wherein the ErbB3 ligand is HRG-β or a functionalfragment thereof.
 6. The method of claim 5, wherein the cell mediumfurther comprising transforming growth factor beta (TGF-β), a TGF-βfamily member or a functional fragment thereof.
 7. The method of claim6, wherein the TGF-β family member is selected from the group consistingof Nodal, Activin A, Activin B, bone morphogenic protein-2 (BMP2), bonemorphogenic protein-4 (BMP4), and functional fragments thereof.
 8. Themethod of claim 7, where the cell medium further comprises Activin A. 9.The method of claim 8, wherein the cell medium further comprisesinsulin-like growth factor or a functional fragment thereof.
 10. Themethod of claim 9, wherein the cell medium is free of exogenousfibroblast growth factor.
 11. The method of claim 9, wherein the cellmedium further comprises at least one fibroblast growth factor (FGF)selected from the group consisting of FGF-2, FGF-7, FGF-10, FGF-22 andvariants functional fragments thereof.
 12. The method of claim 11,wherein the at least one FGF is FGF-7, FGF-10 and FGF-22.
 13. The methodof claim 12, wherein the cell medium comprises a serum albumin (SA). 14.The method of claim 13, wherein the SA is bovine SA (BSA) or human SA(HSA).
 15. The method of claim 14, wherein the concentration of the SAis more than about 0.2%, volume to volume (v/v).
 16. The method of claim14, wherein the concentration of SA is less than about 5% v/v.
 17. Themethod of claim 1, wherein the differentiable cells are primateembryonic stem cells.
 18. The method of claim 1, wherein the cells aremaintained in suspension and in appropriate culture conditions for atleast two days.
 19. A method of expanding differentiable cells insuspension, said method comprising a) placing the differentiable cellsin a suspension of cell medium, the medium comprising a basal saltnutrient solution and being essentially free of serum, and providing ameans for stimulating ErbB2-directed tyrosine kinase activity in thedifferentiable cells; and b) maintaining the differentiable cells in thesuspension of culture medium and in appropriate culture conditions formore than one day, wherein the cell medium is changed periodically,wherein the differentiable cells expand and do not differentiate. 20.The method of claim 18, wherein the means for stimulating ErbB2-directedtyrosine kinase activity comprises at least one ligand that specificallybinds ErbB3 in said differentiable cells.
 21. The method of claim 19,wherein the differentiable cells are primate embryonic stem cells. 22.The method of claim 20, wherein neither insulin nor an insulinsubstitute is provided to said embryonic stem cells.
 23. The method ofclaim 20, further comprising providing insulin-like growth factor or afunctional fragment thereof to said embryonic stem cells.
 24. The methodof claim 22, wherein said ligand that specifically binds ErbB3 isselected from the group consisting of Neuregulin-1, Heregulin-β (HRG-β),Heregulin-α (HRG-α), Neu differentiation factor (NDF), acetylcholinereceptor-inducing activity (ARIA), glial growth factor 2 (GGF2),motor-neuron derived factor (SMDF), Neuregulin-2, Neuregulin-2β(NRG2-β), Epiregulin, Biregulin and functional fragments thereof. 25.The method of claim 23, wherein said ligand is HRG-β or a functionalfragment thereof.
 26. The method of claim 24, further comprisingproviding to said cells transforming growth factor beta (TGF-β), a TGF-βfamily member or a functional fragment thereof.
 27. The method of claim25, wherein said TGF-β family member is selected from the groupconsisting of Nodal, Activin A, Activin B, bone morphogenic protein-2(BMP2), bone morphogenic protein-4 (BMP4), and functional fragmentsthereof.
 28. The method of claim 26, further comprising providinginsulin-like growth factor or a functional fragment thereof to saidembryonic stem cells.
 29. The method of claim 27, further comprisingproviding at least one fibroblast growth factor (FGF) selected from thegroup consisting of FGF-2, FGF-7, FGF-10, FGF-22 and functionalfragments thereof to said embryonic stem cells.
 30. The method of claim28, wherein said at least one FGF is FGF-7, FGF-10 and FGF-22.
 31. Themethod of claim 29, further comprising providing a serum albumin (SA) tosaid embryonic stem cells.
 32. The method of claim 30, wherein the SA isbovine SA (BSA) or human SA (HSA).
 33. The method of claim 31, whereinthe concentration of the SA is more than about 0.2%, volume to volume(v/v).
 34. The method of claim 32, wherein the concentration of SA isless than about 5% v/v.